FN14 binding proteins and uses thereof

ABSTRACT

The present disclosure provides proteins comprising antibody antigen binding domains that bind to Fn14 and uses thereof. The present disclosure also provides methods for treating wasting disorders, such as cachexia.

RELATED APPLICATION DATA

This application is a continuation application of PCT Application No.PCT/AU2012/000989 entitled “FN14 Binding Proteins and Uses Thereof”,filed Aug. 23, 2012 which claims priority to U.S. Patent Application No.61/526,599 entitled “FN14 Binding Proteins and Uses Thereof” filed onAug. 23, 2011. The entire contents of both applications are herebyincorporated by reference.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicform. The entire contents of the Sequence Listing is hereby incorporatedby reference.

FIELD

The present disclosure relates to Fn14-binding proteins comprisingantigen binding domains of anti-Fn14 antibodies and uses thereof andmethods of treating, preventing, diagnosing or prognosing variousconditions including wasting disorders, such as cachexia.

BACKGROUND

Fibroblast Growth Factor Inducible 14

Fibroblast growth factor inducible 14 (Fn14, also known as TNF-like weakinducer of apoptosis receptor [Tweak-R] or TNFRSF12A), is a member ofthe Tumor Necrosis Factor receptor superfamily. Expression of Fn14 isup-regulated by growth factors in vitro and in vivo in response totissue injury, regeneration, and inflammation. As one of the names forFn14 suggests, this protein is a receptor for the protein designatedTweak. Tweak binding to Fn14, or constitutive Fn14 overexpression,activates the NFκB signaling pathway, which is known to play animportant role in immune and inflammatory processes, oncogenesis, andcancer therapy resistance. This interaction also controls many cellularactivities including, proliferation, migration, differentiation,apoptosis, angiogenesis and inflammation. Tweak and Fn14 are alsoinvolved in tissue repair and regulation of immune functions and tumorgrowth. Accordingly, Fn14-mediated signaling is involved in pathwaysthat play important roles in human diseases. Fn14-mediated signaling hasbeen suggested to play a role in numerous diseases, including, cancer,metastasis, immunological disorders (including autoimmune diseases,graft rejection and graft versus host disease, and chronic and acuteneurological conditions [including stroke]).

Fn14 is expressed by many non-lymphoid cell types (epithelial,mesenchymal, endothelial cells and neurons), by many tissue progenitorcells, including all progenitor cells of the mesenchymal lineage. Thisprotein is highly inducible by growth factors e.g., in serum that areencountered in vivo at sites of tissue injuries and/or tissueremodeling. As a consequence Fn14 expression is relatively low in mosthealthy tissues, but increased in injured and/or diseased tissues.

Based on the foregoing description, the skilled artisan will be awarethat compounds that bind to Fn14 are desirable. These compounds can beused to treat, prevent, diagnose or prognose Fn14-mediated conditions.In addition, it is desirable to identify and further understand the roleof Fn14 in various biological conditions and diseases. In particular,the role of Fn14 in wasting disorders has not been previouslydemonstrated.

Wasting Disorders

Wasting disorders are a class of disorders that are characterized byprogressive loss of one or more tissues, e.g., muscle and/or fat.Wasting disorders can be classified into two types: (i) those in whichthe tissue that is lost is the site of the disorder (e.g., musculardystrophy); and (ii) wasting disorders that are associated with orcaused by other conditions. This latter type can be associated with avariety of conditions including motor neuron disease, denervation, andageing. Wasting disorders associated with other disorders also includesa subclass of wasting disorders known as cachexia.

Cachexia is a metabolic disorder associated with underlying illness(i.e., a condition) and characterized by loss of body weight and loss ofmuscle with or without loss of fat mass. Cachexia is generallyassociated with increased protein catabolism due to underlyingdisease(s). Contributory factors to the onset of cachexia are anorexiaand metabolic alterations (e.g., increased inflammatory status,increased muscle proteolysis and impaired carbohydrate, protein andlipid metabolism). Conditions associated with cachexia include, but arenot limited to, cancer, certain infectious diseases (e.g. tuberculosis,AIDS), some autoimmune disorders (including diabetic neuropathiccachexia, and rheumatoid cachexia), or addiction to drugs such asamphetamines or cocaine, chronic alcoholism and cirrhosis of the liver.Cachexia physically weakens patients to a state of immobility stemmingfrom loss of appetite, asthenia, and anemia, and response to standardtreatment is usually poor. Cachexia is regularly seen associated withcancer, and in that context is called “cancer cachexia”. There are alsoa cachectic syndromes observed in patients suffering from disorders,such as, but not limited to renal disease, congestive heart failure,chronic obstructive pulmonary disease, diabetes and some severe cases ofschizophrenia can present this disorder where it is named vesaniccachexia.

A common form of cachexia is associated with cancer. In this regard,cachexia is one of the most common manifestations of cancer and ispresent in up to 80% of patients with advanced cancer, including, butnot limited to cancers of the breast, lung, colon, prostate, pancreasand gastrointestinal tract. Cachexia is also present early inprogression of cancer with, for example, 85% of patients withgastrointestinal cancers, 83% of patients with pancreatic cancer and 60%of patients with lung cancer presenting with cachexia upon diagnosis.Cachexia accounts for >20% of all cancer-related deaths and isassociated with reduced mobility, increased risk of complications insurgery, impaired response to chemo-/radio-therapy, decreased survivaltime and increased psychological distress, leading to an overallreduction in quality of life for sufferers. Currently, the onlydefinitive treatment of cancer cachexia is removal of the causativetumor. Short of achieving this goal, which is often compromised by thepatients' inability to tolerate cancer treatments due to their cachexia,various measures have been undertaken to ameliorate cachexia, howeverwith limited success. Various agents have been administered in attemptsto retard or halt progressive cachexia in cancer patients. These agentsinclude orexigenic agents (appetite stimulants), corticosteroids,cannabinoids, serotonin antagonists, prokinetic agents, androgens andanabolic agents, anticytokine agents, non-steroidal anti-inflammatorydrugs, and regulators of circadian rhythm.

It will be apparent to the skilled artisan from the foregoing that theidentification of improved Fn14-binding proteins for medical anddiagnostic treatment would be useful. Furthermore, the treatment ofwasting disorders, such as cachexia, is a significant unmet medicalneed. Thus, there is a need in the art for therapies that canameliorate, delay or prevent wasting disorders, including, unintendedbody weight loss, cachexia, muscle and/or fat wasting, weakness and/orfatigue associated with one or more disorders. Desirably, such therapiesreduce mortality and/or enhance and/or prolong patients' quality oflife, which can assist with therapeutic and/or prophylactic methods,e.g., to treat a condition causing or associated with the wastingdisorder.

SUMMARY

The inventors have produced a class of antibodies that bind specificallyto Fn14 and reduce Tweak-induced NFκB-signaling in a cell expressingFn14. Accordingly, these antibodies are antagonists of a Tweak-mediatedactivity through Fn14. Antibodies identified by the inventors arecapable of antagonizing or reducing several Tweak-mediated activities(e.g., NFκB-signaling and/or Tweak-induced Kym1 cell death). Antibodiesidentified by the inventors do not induce NFκB-signaling when bound toFn14, e.g., in the absence of Tweak, meaning that these antibodies couldavoid undesirable side-effects in situations in which treatment ofTweak-mediated conditions using antibodies against Fn14 is desirable.This functional difference not only provides a practical benefit, but italso distinguishes the antibodies produced by the inventors from severalknown anti-Fn14 antibodies that may suffer from undesirable side effects(e.g., ITEM-1, ITEM-2 and ITEM-3 and derivatives thereof).

Some antibodies identified by the inventors are capable of inducing oragonizing some Tweak-mediated activities, such as cytokine secretion(e.g., interleukin (IL)-8 secretion).

The inventors have also determined that the antibodies bind to anepitope, e.g., a conformational epitope, contained within a commonregion of the extracellular domain of Fn14. Moreover, the inventorsfound that different residues within the epitope were involved inbinding of different sub-classes of antibodies. In conducting theseanalyses, the inventors determined that they could distinguishantibodies particularly effective in treating wasting conditions (asdiscussed below) from antibodies that were ineffective or less effectivebased on the amino acid residues involved within the epitope in antibodybinding.

In developing animal models to characterize the antibodies that theyproduced, the inventors were surprised to find that mice administeredtumorigenic cells ectopically expressing Fn14 developed severe cachexia.The inventors also determined that a class of antibodies that modulatedFn14 activity is capable of treating cachexia, including inducing weightgain in a subject following progression of cachexia. Upon furthercharacterization of this finding, the inventors determined that theantibodies were exerting a therapeutic effect by binding to andmodulating Fn14 signaling on the tumor cells. Thus, by modulatingFn14-signaling on a tumor, the inventors were inducing a positive effectat a distinct tissue, e.g., skeletal muscle.

The inventors have extended these studies to show that anti-Fn14antibodies they produced were capable of preventing or delaying cachexiain a colon cancer mouse model. An antibody tested by the inventors wasalso able to reduce the size of tumors in the mice.

The inventors additionally demonstrated that a class of antibodies wascapable of extending the life of a subject suffering from cancer and/orcachexia.

The inventors have also shown that an antibody that they have producedprevents a cancer's ability to invade tissue, such as muscle, indicatingutility in preventing tissue invasion or metastasis of cancer.

The inventors further extended their studies by showing that they couldreduce or prevent muscle wasting in a mouse model of diabetes-inducedwasting/cachexia. These data indicate that the inventors have producedantibodies useful for the treatment or prevention of a variety ofwasting disorders.

When treating mice suffering from diabetes using an antibody, theinventors observed that the mice showed reduced water intake and bloodglucose levels compared to untreated diabetic mice.

Thus, the present disclosure provides various reagents fordiagnosing/prognosing/treating/preventing Fn14-mediated conditions,including wasting conditions, such as cachexia. The present disclosurealso provides methods for treating or preventing wasting conditions,such as cachexia or tissue invasion or metastasis of cancer.

In one example, the present disclosure provides an Fn14-binding proteincomprising an antigen binding domain, wherein the antigen binding domainbinds specifically to Fn14 and wherein the protein antagonizes at leastone Tweak-mediated Fn14 signaling effect, and wherein the protein doesnot detectably induce the Tweak-mediated Fn14 signaling effect whencontacted to a cell expressing Fn14 in the absence of Tweak. In oneexample, the antigen binding domain is from or derived from anon-antibody Fn14 binding protein.

In one example, the present disclosure provides an Fn14-binding proteincomprising an antigen binding domain of an anti-Fn14 antibody, whereinthe antigen binding domain binds specifically to Fn14 and wherein theprotein antagonizes at least one Tweak-mediated Fn14 signaling effect,and wherein the protein does not detectably induce the Tweak-mediatedFn14 signaling effect when contacted to a cell expressing Fn14 in theabsence of Tweak.

In one example, the Tweak-mediated activity is Tweak-inducedNFκB-signaling in a cell and/or Tweak-induced death of Kym1 cells.

In one example, the present disclosure provides an Fn14-binding proteincomprising an antigen binding domain, wherein the antigen binding domainbinds specifically to Fn14 and wherein the Fn14-binding protein reducesTweak-induced NFκB-signaling in a cell expressing Fn14, and wherein theFn14-binding protein does not detectably induce NFκB-signaling whencontacted to a cell expressing Fn14 in the absence of Tweak.

In one example, the present disclosure provides an Fn14-binding proteincomprising an antigen binding domain of an anti-Fn14 antibody, whereinthe antigen binding domain binds specifically to Fn14 and wherein theFn14-binding protein reduces Tweak-induced NFκB-signaling in a cellexpressing Fn14, and wherein the Fn14-binding protein does notdetectably induce NFκB-signaling when contacted to a cell expressingFn14 in the absence of Tweak.

For example, NFκB-signaling is detected in a cell (e.g., a HEK293T cell)comprising a nucleic acid encoding fluorescent protein operably linkedto a promoter that facilitates gene expression as a result of NFκBbinding. For example, NFκB signaling is detected using fluorescenceactivated cell sorting. Tweak-induced NFκB-signaling can be induced bycontacting the cell with Tweak (e.g., about 100 ng or 200 ng of Tweak)either in the presence or absence of the protein. In one example, theFn14-binding protein reduces or prevents Tweak-induced NFκB-signaling(e.g., induced by 100 ng or 200 ng of Tweak) at a concentration of 100ng/ml or 1 μg/ml.

For example, the Fn14-binding protein binds specifically to an epitopein Fn14 comprising residues contained within the sequence set forth inSEQ ID NO: 34.

In one example, the Fn14-binding protein does not detectably bind to apolypeptide comprising a region of Fn14, the region consisting of asequence set forth in SEQ ID NO: 33 or 46. For example, the polypeptideis displayed on the surface of a phage.

In one example, the Fn14-binding protein does not detectably bind to apeptide consisting of a sequence set forth in SEQ ID NO: 33 or 46.

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising a proline or alanine substituted for thearginine at position 56 of SEQ ID NO: 1. In one example, theFn14-binding protein binds to an extracellular domain of Fn14 comprisinga proline or alanine substituted for the arginine at position 56 of SEQID NO: 1 at a similar or substantially the same level as it binds to anextracellular domain of Fn14 (e.g., comprising a sequence set forth inSEQ ID NO: 2). For example, the Fn14-binding protein binds to a peptideconsisting of the sequence set forth in SEQ ID NO: 52 or 54 optionallyadditionally comprising six histidine residues at a terminus, e.g., at asimilar or substantially the same level as it binds to an extracellulardomain of Fn14 (e.g., comprising a sequence set forth in SEQ ID NO: 2).In one example, the level of binding is assessed by immobilizing thepeptide and contacting the peptide with the Fn14-binding protein.Exemplary Fn14-binding proteins described herein having such bindingcharacteristics comprise the variable regions and/or CDRs of an antibodydesignated CRCBT-06-001, CRCBT-06-002 or CRCBT-06-005. Thus, in oneexample, the Fn14-binding protein binds to a peptide consisting of thesequence set forth in SEQ ID NO: 52 or 54 (optionally with an additionalsix histidine residues at a terminus) at a similar or substantially thesame level or with a similar or substantially the same affinity as anantibody designated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005. Inanother example, the Fn14-binding protein competitively inhibits bindingof an antibody designated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005to a peptide consisting of the sequence set forth in SEQ ID NO: 52 or 54(optionally with an additional six histidine residues at a terminus).

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising an alanine substituted for the arginine atposition 58 of SEQ ID NO: 1. In one example, the Fn14-binding proteinbinds to an extracellular domain of Fn14 comprising an alaninesubstituted for the arginine at position 58 of SEQ ID NO: 1 at a similaror substantially the same level as it binds to an extracellular domainof Fn14 (e.g., comprising a sequence set forth in SEQ ID NO: 2). Forexample, the Fn14-binding protein binds to a peptide consisting of thesequence set forth in SEQ ID NO: 56 optionally additionally comprisingsix histidine residues at a terminus, e.g., at a similar orsubstantially the same level as it binds to an extracellular domain ofFn14 (e.g., comprising a sequence set forth in SEQ ID NO: 2). In oneexample, the level of binding is assessed by immobilizing the peptideand contacting the peptide with the Fn14-binding protein or byexpressing the peptide on the surface of a phage and contacting thephage to an immobilized Fn14 binding protein. Exemplary Fn14-bindingproteins described herein having such binding characteristics comprisethe variable regions and/or CDRs of an antibody designated CRCBT-06-001,CRCBT-06-002 or CRCBT-06-005. Thus, in one example, the Fn14-bindingprotein binds to a peptide consisting of the sequence set forth in SEQID NO: 56 (optionally with an additional six histidine residues at aterminus) at a similar or substantially the same level or with a similaror substantially the same affinity as an antibody designatedCRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005. In another example, theFn14-binding protein competitively inhibits binding of an antibodydesignated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005 to a peptideconsisting of the sequence set forth in SEQ ID NO: 56 (optionally withan additional six histidine residues at a terminus).

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising an alanine substituted for the histidine atposition 60 of SEQ ID NO: 1. In one example, the Fn14-binding proteinbinds to an extracellular domain of Fn14 comprising an alaninesubstituted for the histidine at position 60 of SEQ ID NO: 1 at asimilar or substantially the same level as it binds to an extracellulardomain of Fn14 (e.g., comprising a sequence set forth in SEQ ID NO: 2).For example, the Fn14-binding protein binds to a peptide consisting ofthe sequence set forth in SEQ ID NO: 63 optionally additionallycomprising six histidine residues at a terminus, e.g., at a similar orsubstantially the same level as it binds to an extracellular domain ofFn14 (e.g., comprising a sequence set forth in SEQ ID NO: 2). In oneexample, the level of binding is assessed by immobilizing the peptideand contacting the peptide with the Fn14-binding protein. ExemplaryFn14-binding proteins described herein having such bindingcharacteristics comprise the variable regions and/or CDRs of an antibodydesignated CRCBT-06-001 or CRCBT-06-002. Thus, in one example, theFn14-binding protein binds to a peptide consisting of the sequence setforth in SEQ ID NO: 63 (optionally with an additional six histidineresidues at a terminus) at a similar or substantially the same level orwith a similar or substantially the same affinity as an antibodydesignated CRCBT-06-001 or CRCBT-06-002. In another example, theFn14-binding protein competitively inhibits binding of an antibodydesignated CRCBT-06-001 or CRCBT-06-002 to a peptide consisting of thesequence set forth in SEQ ID NO: 63 (optionally with an additional sixhistidine residues at a terminus).

In one example, the Fn14-binding protein binds to a peptide consistingof the sequence set forth in SEQ ID NO: 48 (optionally with anadditional six histidine residues at a terminus). In one example, theFn14-binding protein binds to a peptide consisting of the sequence setforth in SEQ ID NO: 48 (optionally with an additional six histidineresidues at a terminus) at a similar or substantially the same level asit binds to an extracellular domain of Fn14 (e.g., comprising a sequenceset forth in SEQ ID NO: 2). In one example, the level of binding isassessed by immobilizing the peptide and contacting the peptide with theFn14-binding protein. Exemplary Fn14-binding proteins described hereinhaving such binding characteristics comprise the variable regions and/orCDRs of an antibody designated CRCBT-06-001 or CRCBT-06-002. Thus, inone example, the Fn14-binding protein binds to a peptide consisting ofthe sequence set forth in SEQ ID NO: 48 (optionally with an additionalsix histidine residues at a terminus) at a similar or substantially thesame level or with a similar or substantially the same affinity as anantibody designated CRCBT-06-001 or CRCBT-06-002. In another example,the Fn14-binding protein competitively inhibits binding of an antibodydesignated CRCBT-06-001 or CRCBT-06-002 to a peptide consisting of thesequence set forth in SEQ ID NO: 48 (optionally with an additional sixhistidine residues at a terminus).

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising an alanine substituted for the tryptophan atposition 42 of SEQ ID NO: 1. In one example, the Fn14-binding proteinbinds to an extracellular domain of Fn14 comprising an alaninesubstituted for the tryptophan at position 42 of SEQ ID NO: 1 at asimilar or substantially the same level as it binds to an extracellulardomain of Fn14 (e.g., comprising a sequence set forth in SEQ ID NO: 2).For example, the Fn14-binding protein binds to a peptide consisting ofthe sequence set forth in SEQ ID NO: 57 optionally additionallycomprising six histidine residues at a terminus, e.g., at a similar orsubstantially the same level as it binds to an extracellular domain ofFn14 (e.g., comprising a sequence set forth in SEQ ID NO: 2). In oneexample, the level of binding is assessed by immobilizing the peptideand contacting the peptide with the Fn14-binding protein. ExemplaryFn14-binding proteins described herein having such bindingcharacteristics comprise the variable regions and/or CDRs of an antibodydesignated CRCBT-06-001, CRCBT-06-002 or CRCBT-06-004. Thus, in oneexample, the Fn14-binding protein binds to a peptide consisting of thesequence set forth in SEQ ID NO: 57 (optionally with an additional sixhistidine residues at a terminus) at a similar or substantially the samelevel or with a similar or substantially the same affinity as anantibody designated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-004. Inanother example, the Fn14-binding protein competitively inhibits bindingof an antibody designated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-004to a peptide consisting of the sequence set forth in SEQ ID NO: 57(optionally with an additional six histidine residues at a terminus).

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising one of the following amino acid substitutions(numbering relative to SEQ ID NO: 1) T33N, A34S, R38S, R56P, L77M, R56A,R56K, R58A, W42A, D51A, S54A, A57G, P59A, H60A, S61A, D62A, F63A, L65Aor H60K. In one example, the level of binding is similar orsubstantially the same level as the Fn14-binding protein binds to anextracellular domain of Fn14 (e.g., comprising a sequence set forth inSEQ ID NO: 2).

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising one or more amino acid substitutionsindividually or collectively selected from the group consisting of(numbering relative to SEQ ID NO: 1) T33N, A34S, R38S, R56P, L77M, R56A,R56K, R58A, W42A, L46A, D51A, S54A, A57G, P59A, H60A, S61A, D62A, F63A,L65A or H60K. In one example, the level of binding is similar orsubstantially the same level as the Fn14-binding protein binds to anextracellular domain of Fn14 (e.g., comprising a sequence set forth inSEQ ID NO: 2).

In one example, the Fn14 binding protein is capable of binding to apeptide consisting of a sequence set forth in any one of SEQ ID NOs:49-68, optionally with an additional six histidine residues at aterminus.

In one example, the Fn14-binding protein is capable of binding to aseries of peptides, wherein the series comprises peptides having all ofthe sequences set forth in any one of SEQ ID NOs: 49-68, optionally withan additional six histidine residues at a terminus.

In one example, the Fn14 binding protein is capable of binding to apeptide consisting of a sequence set forth in SEQ ID NOs: 47, optionallywith an additional six histidine residues at a terminus.

In one example, the Fn14 binding protein is capable of binding to apeptide consisting of a sequence set forth in SEQ ID NO: 48, optionallywith an additional six histidine residues at a terminus.

In one example, the Fn14-binding protein binds to a peptide consistingof a sequence set forth in SEQ ID NO: 35 in an amount within 75% of theamount of bound by an antibody comprising a V_(H) comprising a sequenceset forth in SEQ ID NO: 19 and a V_(L) comprising a sequence set forthin SEQ ID NO: 26. In one example, the amount of protein or antibodybound is assessed by contacting the Fn14-binding protein to a peptideconsisting of the sequence set forth in SEQ ID NO: 35 and an amount ofthe Fn14-binding protein (e.g., 2 μg/ml) brought into contact with thepeptide. The amount of Fn14-binding protein bound to the peptide is thendetermined and compared to the amount of an antibody comprising a V_(H)comprising a sequence set forth in SEQ ID NO: 19 and a V_(L) comprisinga sequence set forth in SEQ ID NO: 26 bound to the peptide. In oneexample, the amount of Fn14-binding protein bound to the peptide iswithin about 73% or 60% or 45% of the amount of antibody bound.

In one example, the Fn14-binding protein binds to a peptide consistingof a sequence set forth in SEQ ID NO: 34 in an amount within 80% of theamount bound by an antibody comprising a V_(H) comprising a sequence setforth in SEQ ID NO: 15 and a V_(L) comprising a sequence set forth inSEQ ID NO: 22 or comprising a V_(H) comprising a sequence set forth inSEQ ID NO: 16 and a V_(L) comprising a sequence set forth in SEQ ID NO:23. In one example, the amount of protein or antibody bound is assessedby contacting the Fn14-binding protein to a peptide consisting of thesequence set forth in SEQ ID NO: 34 and an amount of the Fn14-bindingprotein (e.g., 5 ng/ml) brought into contact with the peptide. Theamount of Fn14-binding protein bound to the peptide is then determinedand compared to the amount of the antibody bound to the peptide. In oneexample, the amount of Fn14-binding protein bound to the peptide iswithin about 50% or 48% or 45% or 40% of the amount of antibody bound.

The present disclosure also provides Fn14-binding protein comprising anantigen binding domain of an anti-Fn14 antibody, wherein the antigenbinding domain binds specifically to a peptide comprising residues of anepitope bound by antibodies identified by the inventors. For example,the disclosure provides an Fn14-binding protein comprising an antigenbinding domain of an of an anti-Fn14 antibody, wherein the antigenbinding domain binds specifically to Fn14, wherein the antigen bindingdomain binds to an epitope in Fn14 comprising residues contained withinthe sequence set forth in SEQ ID NO: 34.

For example, the antigen binding domain binds specifically to an epitopein Fn14 comprising residues contained within one or more sequences setforth in SEQ ID NOs: 3 to 9 or 34.

In one example, the Fn14-binding protein binds to a conformationalepitope in Fn14. For example, the conformational epitope is dependent ondisulphide bond formation within Fn14. For example, the conformationalepitope bound by the Fn14-binding protein is not present in an Fn14protein lacking disulphide bonds, e.g., Fn14 in reduced form.

In one example, the Fn14-binding protein does not substantially bind toFn14 in reduced form or reduced and alkylated form. In one example, theFn14-binding protein does not substantially bind to a polypeptidecomprising an extracellular domain of Fn14 fused to an Fc region of anantibody, wherein the polypeptide is reduced or reduced and alkylated.For example, the binding is detected by performing an enzyme-linkedimmunosorbent assay (ELISA) to detect binding of the Fn14-bindingprotein to the immobilized polypeptide.

For example, the Fn14-binding protein does not detectably bind to Fn14in reduced form or reduced and alkylated form. In one example, theFn14-binding protein does not detectably bind to a polypeptidecomprising an extracellular domain of Fn14 fused to an Fc region of anantibody, wherein the polypeptide is reduced or reduced and alkylated.

In one example, the Fn14-binding protein reduces Tweak-inducedinterleukin-8 (IL-8) secretion from a cell (i.e., has IL-8 secretionantagonistic activity), e.g., an A375 human melanoma cell. In oneexample, the Fn14-binding protein (e.g., at a concentration of about 10μg/ml) reduces IL-8 secretion by A375 melanoma cells (e.g., about 1×10⁴cells) contacted with Tweak fused to the Fc region of an antibody (e.g.,at a concentration of about 300 ng/ml). An exemplary antibody havingsuch activity comprises the variable regions or CDRs of an antibodyselected from the group consisting of CRCBT-06-002, CRCBT-06-005 andCRCBT-06-007 or antibodies comprising CDRs thereof.

In the case of CRCBT-06-005, the antibody is, for example, mouse IgG2b(the equivalent of human IgG3). In this regard, the inventors have shownthat CRCBT-06-005 and CRCBT-06-006 have very similar V regions, howeverdiffer in isotype and activity (e.g., CRCBT-06-006 [mouse IgG2a,equivalent of human IgG1] is an agonist of Tweak-induced IL-8secretion).

In one example, the Fn14-binding protein has a level of antagonism of atleast about 80% or 90% or 100% when assessed using the followingequation (b−d)/(b−a), where a=the amount of IL-8 secreted in thepresence of 10 μg/ml antibody, b=the amount of IL-8 secreted in thepresence of 300 ng/ml Tweak-Fc, c=the amount of IL-8 secreted from cellsin the absence of either antibody or Tweak-Fc, d=the amount of IL-8secreted in the presence of 10 μg/ml antibody and 300 ng/ml Tweak-Fc.

In one example, the Fn14-binding protein induces Tweak-inducedinterleukin-8 (IL-8) secretion from a cell (i.e., has IL-8 secretionagonistic activity), e.g., an A375 human melanoma cell when the cell iscontacted with the Fn14-binding protein in the absence of Tweak. In oneexample, the Fn14-binding protein (e.g., at a concentration of about 10,1, 0.1 or 0.01 mg/ml) induces IL-8 secretion by A375 melanoma cells(e.g., about 1×10⁴ cells) in the absence of Tweak. An exemplary antibodyhaving such activity comprises the variable regions or CDRs of anantibody selected from the group consisting of CRCBT-06-001 andCRCBT-06-006.

In one example, the Fn14-binding protein has a level of agonism of atleast about 80% or 90% when assessed using the following equation(a−c)/(b−c), where a=the amount of IL-8 secreted in the presence of 10μg/ml antibody, b=the amount of IL-8 secreted in the presence of 300ng/ml Tweak-Fc, c=the amount of IL-8 secreted from cells in the absenceof either antibody or Tweak-Fc, d=the amount of IL-8 secreted in thepresence of 10 μg/ml antibody and 300 ng/ml Tweak-Fc.

In one example, the Fn14-binding protein delays or prevents weight lossupon administration to a subject suffering from cancer orcancer-cachexia or diabetes or diabetes-induced cachexia.

In one example, the Fn14-binding protein binds to a protein comprisingan extracellular domain of Fn14 fused to an Fc region of an antibodywith an affinity dissociation constant (K_(D)) of 2 nM or less, such as,1.5 nM or less, for example, 1 nM or less. In one example, the K_(D) isassessed by immobilizing the Fn14-binding protein (e.g., a Fab or anantibody) and assessing binding of an extracellular region of Fn14 fusedto an Fc to the immobilized Fn14-binding protein using surface plasmonresonance.

In one example, the K_(D) is between about 0.01 nM to about 2 nM, suchas between about 0.05 nM to about 1 nM, for example, between about 0.1nM to about 1 nM, for example, between about 0.5 nM to about 1 nM. Inone example, the K_(D) is assessed by immobilizing the Fn14-bindingprotein (e.g., a Fab or an antibody) and assessing binding of anextracellular region of Fn14 fused to an Fc to the immobilizedFn14-binding protein using surface plasmon resonance.

An exemplary Fn14-binding protein of the disclosure has a K_(D) of about0.9 nM (e.g., +/−0.1 nM) for a protein comprising an extracellulardomain of Fn14 fused to an Fc region of an antibody. Another exemplaryFn14-binding protein of the disclosure has a K_(D) of about 0.7 nM(e.g., +/−0.1 nM) for a protein comprising an extracellular domain ofFn14 fused to an Fc region of an antibody. In one example, theFn14-binding protein binds to recombinant human Fn14 with a K_(D) of 0.6nM or less.

In one example, the Fn14-binding protein binds to recombinant human Fn14with a K_(D) of 1 nM or less, such as, 0.9 nM or less, for example, 0.8nM or less, for example, 0.7 nM or less, for example, 0.6 nM or less. Inone example, the K_(D) is assessed by immobilizing the Fn14-bindingprotein (e.g., a Fab or an antibody) and assessing binding of humanrecombinant Fn14 to the immobilized Fn14-binding protein using surfaceplasmon resonance.

In one example, the K_(D) is between about 0.01 nM and 1 nM, such asbetween about 0.05 nM and 0.9 nM, for example, between about 0.09 nM and0.7 nM, for example, between about 0.1 nM and 0.6 nM.

An exemplary Fn14-binding protein of the disclosure has a K_(D) of about0.5 nM (e.g., +/−0.1 nM) for recombinant human Fn14. For example, theFn-14 binding protein is CRCBT-06-001 or an antibody comprising theantigen binding domain, variable regions or CDRs thereof.

Another exemplary Fn14-binding protein of the disclosure has a K_(D) ofabout 0.2 nM (e.g., +/−0.1 nM) for recombinant human Fn14. For example,the Fn-14 binding protein is CRCBT-06-002 or an antibody comprising theantigen binding domain, variable regions or CDRs thereof.

In one example, the Fn14-binding protein binds to a protein comprisingan extracellular domain of Fn14 fused to an Fc region of an antibodywith an on-rate (Ka) of 5×10³M⁻¹s⁻¹ or greater, such as about2×10³M⁻¹s⁻¹ or greater, for example, about 1.5×10⁴M⁻¹s⁻¹ or greater. Inone example, the Ka is assessed by immobilizing the Fn14-binding protein(e.g., a Fab or an antibody) and assessing binding of an extracellularregion of Fn14 fused to an Fc to the immobilized Fn14-binding proteinusing surface plasmon resonance.

For example, the Ka is between about 5×10³M⁻¹s⁻¹ to about 5×10⁵M⁻¹s⁻¹,for example, between about 1×10⁴M⁻¹s⁻¹ to about 4×10⁵M⁻¹s⁻¹, forexample, between about 2×10⁴M⁻¹s⁻¹ to about 4×10⁵M⁻¹s⁻¹. In one example,the Ka is assessed by immobilizing the Fn14-binding protein (e.g., a Fabor an antibody) and assessing binding of an extracellular region of Fn14fused to an Fc to the immobilized Fn14-binding protein using surfaceplasmon resonance.

An exemplary Fn14-binding protein of the disclosure has a Ka of about2.2×10⁴M⁻¹s⁻¹. A further exemplary Fn14-binding protein of thedisclosure has a Ka of about 3.9×10⁵M⁻¹s⁻¹. In one example, the Ka isassessed by immobilizing the Fn14-binding protein (e.g., a Fab or anantibody) and assessing binding of an extracellular region of Fn14 fusedto an Fc to the immobilized Fn14-binding protein using surface plasmonresonance.

In one example, the Fn14-binding protein binds to recombinant human Fn14with a Ka of 1×10⁵M⁻¹s⁻¹ or greater, such as about 1.2×10⁵M⁻¹s⁻¹ orgreater, for example, about 1.3×10⁵M⁻¹s⁻¹ or greater. In one example,the Ka is assessed by immobilizing the Fn14-binding protein (e.g., a Fabor an antibody) and assessing binding of recombinant human Fn14 to theimmobilized Fn14-binding protein using surface plasmon resonance.

For example, the Ka is between about 1×10⁵M⁻¹s⁻¹ to about 3.5×10⁵M⁻¹s⁻¹.In one example, the Ka is assessed by immobilizing the Fn14-bindingprotein (e.g., a Fab or an antibody) and assessing binding ofrecombinant human Fn14 to the immobilized Fn14-binding protein usingsurface plasmon resonance.

An exemplary Fn14-binding protein of the disclosure has a Ka of about1.8×10⁵M⁻¹s⁻¹ for recombinant human Fn14. For example, the Fn-14 bindingprotein is CRCBT-06-001 or an antibody comprising the antigen bindingdomain, variable regions or CDRs thereof. In one example, the Ka isassessed by immobilizing the Fn14-binding protein (e.g., a Fab or anantibody) and assessing binding of recombinant human Fn14 to theimmobilized Fn14-binding protein using surface plasmon resonance.

A further exemplary Fn14-binding protein of the disclosure has a Ka ofabout 1.3×10⁵M⁻¹s⁻¹ for recombinant human Fn14. For example, the Fn-14binding protein is CRCBT-06-002 or an antibody comprising the antigenbinding domain, variable regions or CDRs thereof. In one example, the Kais assessed by immobilizing the Fn14-binding protein (e.g., a Fab or anantibody) and assessing binding of recombinant human Fn14 to theimmobilized Fn14-binding protein using surface plasmon resonance.

In one example, the Fn14-binding protein binds to a protein comprisingan extracellular domain of Fn14 fused to an Fc region of an antibodywith an off-rate (Kd) of 9×10⁻⁵s⁻¹ or less, such as, 7×10⁻⁵ s⁻¹ or less,for example, 6×10⁻⁵ s⁻¹ or less, for example, 5.5×10⁻⁵ s⁻¹ or less. Inone example, the Kd is assessed by immobilizing the Fn14-binding protein(e.g., a Fab or an antibody) and assessing binding of an extracellularregion of Fn14 fused to an Fc to the immobilized Fn14-binding proteinusing surface plasmon resonance.

For example, the Kd is between about 1×10⁻⁴s⁻¹ to about 1×10⁻⁵s⁻¹, forexample, between about 6×10⁻⁵s⁻¹ to about 1×10⁻⁵s⁻¹, for example,between about 5.5×10⁻⁵s⁻¹ to about 1.4×10⁻⁵s⁻¹, for example, betweenabout 2×10⁻⁵s⁻¹ to about 1.4×10⁻⁵s⁻¹. In one example, the Kd is assessedby immobilizing the Fn14-binding protein (e.g., a Fab or an antibody)and assessing binding of an extracellular region of Fn14 fused to an Fcto the immobilized Fn14-binding protein using surface plasmon resonance.

An exemplary protein of the disclosure has a Kd of about 1.85×10⁻⁵s⁻¹. Afurther exemplary Fn14-binding protein of the disclosure has a Kd ofabout 1.45×10⁻⁵s⁻¹. In one example, the Kd is assessed by immobilizingthe Fn14-binding protein (e.g., a Fab or an antibody) and assessingbinding of an extracellular region of Fn14 fused to an Fc to theimmobilized Fn14-binding protein using surface plasmon resonance.

In one example, the Fn14-binding protein binds to recombinant human Fn14with a Kd of 3×10⁻³s⁻¹ or less.

In one example, the Fn14-binding protein binds to recombinant human Fn14with a Kd of about 8×10⁻⁴s⁻¹ or less, such as, 5×10⁻⁴ s⁻¹ or less, forexample, 4×10⁻⁴ s⁻¹ or less, for example, 2×10⁻⁴ s⁻¹ or less. In oneexample, the Kd is assessed by immobilizing the Fn14-binding protein(e.g., a Fab or an antibody) and assessing binding of recombinant humanFn14 to the immobilized Fn14-binding protein using surface plasmonresonance.

For example, the Kd is between about 9×10⁻⁴s⁻¹ to about 1×10⁻⁵s⁻¹, forexample, between about 5×10⁻⁴s⁻¹ to about 1×10⁻⁵s⁻¹, for example,between about 2×10⁻⁴s⁻¹ to about 2×10⁻⁵s⁻¹. In one example, the Kd isassessed by immobilizing the Fn14-binding protein (e.g., a Fab or anantibody) and assessing binding of recombinant human Fn14 to theimmobilized Fn14-binding protein using surface plasmon resonance.

An exemplary protein of the disclosure has a Kd of about 1.02×10⁻⁴s⁻¹for recombinant human Fn14. For example, the Fn14-binding protein isCRCBT-06-001 or an antibody comprising the antigen binding domain,variable regions or CDRs thereof. In one example, the Kd is assessed byimmobilizing the Fn14-binding protein (e.g., a Fab or an antibody) andassessing binding of recombinant human Fn14 to the immobilizedFn14-binding protein using surface plasmon resonance.

A further exemplary Fn14-binding protein of the disclosure has a Kd ofabout 1.45×10⁻⁵s⁻¹ for recombinant human Fn14. For example, the Fn-14binding protein is CRCBT-06-001 or an antibody comprising the antigenbinding domain, variable regions or CDRs thereof. In one example, the Kdis assessed by immobilizing the Fn14-binding protein (e.g., a Fab or anantibody) and assessing binding of recombinant human Fn14 to theimmobilized Fn14-binding protein using surface plasmon resonance.

In one example, the level of binding of the Fn14-binding protein of thedisclosure to a polypeptide comprising a sequence set forth in any oneof SEQ ID NOs: 29-32 is similar to the level of binding to a polypeptidecomprising a sequence set forth in SEQ ID NO: 2.

In one example, Fn14-binding of the disclosure does not detectably bindto a peptide comprising one or more of the following sequences:

-   (i) an amino acid sequence in SEQ ID NO: 12, wherein the peptide    does not comprise amino acids 1 to 61 of SEQ ID NO: 1;-   (ii) an amino acid sequence set forth in SEQ ID NO: 10; and/or-   (iii) an amino acid sequence set forth in SEQ ID NO: 11.

In one example, an Fn14-binding protein of the disclosure binds to humanFn14 and mouse Fn14. In one example, the binding of the protein isassessed by ELISA using recombinant Fn14 extracellular domain.

In one example, the Fn14-binding protein competitively inhibits bindingof any one of the following antibodies to Fn14 and/or human Fn14 and/ora polypeptide comprising a sequence set forth in SEQ ID NO: 1, 2 or 34:

-   (i) an antibody comprising a heavy chain variable region (V_(H))    comprising a sequence set forth in SEQ ID NO: 15 and a light chain    variable region (V_(L)) comprising a sequence set forth in SEQ ID    NO: 22;-   (ii) an antibody comprising a V_(H) comprising a sequence set forth    in SEQ ID NO: 16 and a V_(L) comprising a sequence set forth in SEQ    ID NO: 23;-   (iii) an antibody comprising a V_(H) comprising a sequence set forth    in SEQ ID NO: 17 and a V_(L) comprising a sequence set forth in SEQ    ID NO: 24;-   (iv) an antibody comprising a V_(H) comprising a sequence set forth    in SEQ ID NO: 18 and a V_(L) comprising a sequence set forth in SEQ    ID NO: 25;-   (v) an antibody comprising a V_(H) comprising a sequence set forth    in SEQ ID NO: 19 and a V_(L) comprising a sequence set forth in SEQ    ID NO: 26;-   (vi) an antibody comprising a V_(H) comprising a sequence set forth    in SEQ ID NO: 20 and a V_(L) comprising a sequence set forth in SEQ    ID NO: 27; and-   (vii) an antibody comprising a V_(H) comprising a sequence set forth    in SEQ ID NO: 21 and a V_(L) comprising a sequence set forth in SEQ    ID NO: 28.

In one example, the Fn14-binding protein binds to the same epitope inFn14 or to an epitope in Fn14 that overlaps with the epitope bound byany one or more of the following antibodies:

-   (i) an antibody comprising a heavy chain variable region (V_(H))    comprising a sequence set forth in SEQ ID NO: 15 and a light chain    variable region (V_(L)) comprising a sequence set forth in SEQ ID    NO: 22;-   (ii) an antibody comprising a V_(H) comprising a sequence set forth    in SEQ ID NO: 16 and a V_(L) comprising a sequence set forth in SEQ    ID NO: 23;-   (iii) an antibody comprising a V_(H) comprising a sequence set forth    in SEQ ID NO: 17 and a V_(L) comprising a sequence set forth in SEQ    ID NO: 24;-   (iv) an antibody comprising a V_(H) comprising a sequence set forth    in SEQ ID NO: 18 and a V_(L) comprising a sequence set forth in SEQ    ID NO: 25;-   (v) an antibody comprising a V_(H) comprising a sequence set forth    in SEQ ID NO: 19 and a V_(L) comprising a sequence set forth in SEQ    ID NO: 26;-   (vi) an antibody comprising a V_(H) comprising a sequence set forth    in SEQ ID NO: 20 and a V_(L) comprising a sequence set forth in SEQ    ID NO: 27; and-   (vii) an antibody comprising a V_(H) comprising a sequence set forth    in SEQ ID NO: 21 and a V_(L) comprising a sequence set forth in SEQ    ID NO: 28.

In one example, the Fn14-binding protein neutralizes a Tweak activity ina cell expressing Fn14. For example, the Fn14-binding protein inhibitsor reduces Tweak-mediated death of Kym1 cells.

For example, at a concentration of about 100 ng/ml, the Fn14-bindingprotein is capable of reducing cell death induced by about 50 ng/ml of aprotein comprising Tweak fused to an Fc region of an antibody. In oneexample, the reduction in cell death is about a 1.5 fold reduction or a2 fold reduction compared to the level of cell death in the presence ofthe same concentration of a protein comprising Tweak fused to an Fcregion of an antibody and the absence of the Fn14-binding protein.

For example, at a concentration of about 1 μg/ml, the Fn14-bindingprotein is capable of reducing cell death induced by about 200 ng/ml ofa protein comprising Tweak fused to an Fc region of an antibody. In oneexample, the reduction in cell death is about a 1.2 fold reduction or a1.5 fold reduction or a 2 fold reduction or a 3 fold reduction comparedto the level of cell death in the presence of the same concentration ofa protein comprising Tweak fused to an Fc region of an antibody and theabsence of the Fn14-binding protein.

Alternatively, or additionally, the Fn14-binding protein reduces orprevents Fn14 (e.g. hFn14)-mediated cytokine production, e.g.,production of IL-6 by tumor cells, e.g., fibroblasts geneticallymodified to express v12Hras and hFn14. In one example, the Fn14-bindingprotein reduces Fn14 (e.g., hFn14)-mediated IL-6 production byfibroblasts genetically modified to express v12Hras and hFn14 (e.g., asdescribed herein) by at least 1.5 fold or at least 2 fold when contactedto the cells at a concentration of about 0.5 μg/ml.

Alternatively, or additionally, the Fn14-binding protein reduces orinhibits Tweak-induced NFκB-signaling in a cell expressing Fn14 (e.g.,hFn14), e.g., as described herein.

In one example, the Fn14-binding protein comprising a non-antibodyantigen binding domain, such as a non-antibody-derived Fn-14 bindingprotein comprising one or more Ig folds (e.g., an immunoglobulin, suchas a T cell receptor or a V domain or an IgNAR), an adnectin, anaffibody, an atrimer, an evasin, a designed ankyrin-repeat protein(DARPin) or an anticalin.

In one example, the Fn14-binding protein comprises an antibody antigenbinding domain.

In one example, the antigen binding domain comprises:

-   (i) a V_(H) comprising a sequence set forth in any one of SEQ ID    NOs: 13 or 15 to 21 or a humanized, chimeric or deimmunized version    thereof; and/or-   (ii) a V_(L) comprising a sequence set forth in any one of SEQ ID    NOs: 14 or 22 to 28 or a humanized, chimeric or deimmunized version    thereof.

In one example, the Fn14-binding protein comprises a V_(H) and a V_(L),wherein the V_(H) and V_(L) bind to form a Fv comprising the antigenbinding domain. For example, the Fv comprises:

-   (i) a V_(H) comprising three complementarity determining regions    (CDRs) of a V_(H) comprising a sequence set forth in SEQ ID NO: 13,    wherein if the amino acid at a position corresponding to residue 101    of SEQ ID NO: 13 is a thymidine, the residue at position 107 is a    histidine and/or the residue at position 53 of SEQ ID NO: 13 is a    serine; and-   (ii) a V_(L) comprising three CDRs of a V_(L) comprising a sequence    set forth in SEQ ID NO: 14.

In one example, the Fv comprises:

-   (i) a V_(H) comprising CDRs 1, 2, and 3 of a V_(H) comprising a    sequence set forth in SEQ ID NO: 15 and a V_(L) comprising CDRs 1,    2, and 3 of a V_(L) comprising a sequence set forth in SEQ ID NO:    22;-   (ii) a V_(H) comprising CDRs 1, 2, and 3 of a V_(H) comprising a    sequence set forth in SEQ ID NO: 16 and a V_(L) comprising CDRs 1,    2, and 3 of a V_(L) comprising a sequence set forth in SEQ ID NO:    23;    (iii) a V_(H) comprising CDRs 1, 2, and 3 of a V_(H) comprising a    sequence set forth in SEQ ID NO: 17 and a V_(L) comprising CDRs 1,    2, and 3 of a V_(L) comprising a sequence set forth in SEQ ID NO:    24;    (iv) a V_(H) comprising CDRs 1, 2, and 3 of a V_(H) comprising a    sequence set forth in SEQ ID NO: 18 and a V_(L) comprising CDRs 1,    2, and 3 of a V_(L) comprising a sequence set forth in SEQ ID NO:    25;-   (v) a V_(H) comprising CDRs 1, 2, and 3 of a V_(H) comprising a    sequence set forth in SEQ ID NO: 19 and a V_(L) comprising CDRs 1,    2, and 3 of a V_(L) comprising a sequence set forth in SEQ ID NO:    26;-   (vi) a V_(H) comprising CDRs 1, 2, and 3 of a V_(H) comprising a    sequence set forth in SEQ ID NO: 20 and a V_(L) comprising CDRs 1,    2, and 3 of a V_(L) comprising a sequence set forth in SEQ ID NO:    27; and-   (vii) a V_(H) comprising CDRs 1, 2, and 3 of a V_(H) comprising a    sequence set forth in SEQ ID NO: 21 and a V_(L) comprising CDRs 1,    2, and 3 of a V_(L) comprising a sequence set forth in SEQ ID NO:    28.

In one example, the CDRs are defined by the Kabat numbering system andare shown in FIGS. 11A-11D in bold text.

In one example, the CDRs are defined by the Chothia numbering system andare shown in FIGS. 11A-11D in underlined text.

In one example, an Fn14-binding protein of the disclosure is a domainantibody (e.g., comprises only a V_(H) or a only a V_(L), for exampleonly a V_(H), optionally linked to another moiety, such as one or moreconstant domains or a constant region or a Fc). In this regard, theinventors have produced CRCBT-06-003 and CRCBT-06-004, which have VLdomains which contain frame-shift mutations meaning that CDRs2 and 3 arenot readily identifiable. Thus, at least the majority of the binding ofthese proteins is expected to result from the V_(H). The sequences ofthe V_(H) domains is very similar to the V_(H) sequences of otherexemplary proteins of the disclosure.

In one example of an Fn14-binding protein of the disclosure, the V_(H)and the V_(L) are in a single polypeptide chain. For example, theFn14-binding protein is:

-   (i) a single chain Fv fragment (scFv);-   (ii) a dimeric scFv (di-scFv);-   (iii) at least one of (i) and/or (ii) linked to a heavy chain    constant region or an Fc or a heavy chain constant domain (C_(H)) 2    and/or C_(H)3; or-   (iv) at least one of (i) and/or (ii) linked to a protein that binds    to an immune effector cell.

In another example, of the disclosure the V_(L) and V_(H) are inseparate polypeptide chains. For example, the Fn14-binding protein is:

-   (i) a diabody;-   (ii) a triabody;-   (iii) a tetrabody;-   (iv) a Fab;-   (v) a F(ab′)₂;-   (vi) a Fv; or-   (vii) at least one of (i) to (vi) linked to a heavy chain constant    region or an Fc or a heavy chain constant domain (C_(H)) 2 and/or    C_(H)3.-   (viii) at least one of (i) to (vi) linked to a protein that binds to    an immune effector cell.

In one exemplary form of the disclosure, the Fn14-binding protein is anantibody.

The present disclosure additionally provides an anti-Fn14 antibody, theantibody comprising any one of the following:

-   (i) a V_(H) comprising a sequence set forth in SEQ ID NO: 13 or a    humanized, synhumanized or deimmunized version thereof and a V_(L)    comprising a sequence set forth in SEQ ID NO: 14 or a humanized,    synhumanized or deimmunized version thereof;-   (ii) a V_(H) comprising a sequence set forth in SEQ ID NO: 15 or a    humanized, synhumanized or deimmunized version thereof and a V_(L)    comprising a sequence set forth in SEQ ID NO: 22 or a humanized,    synhumanized or deimmunized version thereof;-   (iii) a V_(H) comprising a sequence set forth in SEQ ID NO: 16 or a    humanized, synhumanized or deimmunized version thereof and a V_(L)    comprising a sequence set forth in SEQ ID NO: 23 or a humanized,    synhumanized or deimmunized version thereof;-   (iv) a V_(H) comprising a sequence set forth in SEQ ID NO: 17 or a    humanized, synhumanized or deimmunized version thereof and a V_(L)    comprising a sequence set forth in SEQ ID NO: 24 or a humanized,    synhumanized or deimmunized version thereof;-   (v) a V_(H) comprising a sequence set forth in SEQ ID NO: 18 or a    humanized, synhumanized or deimmunized version thereof and a V_(L)    comprising a sequence set forth in SEQ ID NO: 25 or a humanized,    synhumanized or deimmunized version thereof;-   (vi) a V_(H) comprising a sequence set forth in SEQ ID NO: 19 or a    humanized, synhumanized or deimmunized version thereof and a V_(L)    comprising a sequence set forth in SEQ ID NO: 26 or a humanized,    synhumanized or deimmunized version thereof;-   (vii) a V_(H) comprising a sequence set forth in SEQ ID NO: 20 or a    humanized, synhumanized or deimmunized version thereof and a V_(L)    comprising a sequence set forth in SEQ ID NO: 27 or a humanized,    synhumanized or deimmunized version thereof; or    (viii) a V_(H) comprising a sequence set forth in SEQ ID NO: 21 or a    humanized, synhumanized or deimmunized version thereof and a V_(L)    comprising a sequence set forth in SEQ ID NO: 28 or a humanized,    synhumanized or deimmunized version thereof.

The present disclosure additionally provides an anti-Fn14 antibody, theantibody comprising a V_(H) comprising a sequence set forth in SEQ IDNO: 15 or a humanized, synhumanized or deimmunized version thereof and aV_(L) comprising a sequence set forth in SEQ ID NO: 22 or a humanized,synhumanized or deimmunized version thereof.

The present disclosure additionally provides an anti-Fn14 antibody, theantibody comprising a V_(H) comprising a sequence set forth in SEQ IDNO: 16 or a humanized, synhumanized or deimmunized version thereof and aV_(L) comprising a sequence set forth in SEQ ID NO: 23 or a humanized,synhumanized or deimmunized version thereof.

The present disclosure also provides a chimeric antibody comprising aV_(H) and a V_(L) as discussed above (e.g., in the preceding lists),wherein the V_(H) is linked to a human heavy chain constant region andthe V_(L) is linked to a human light chain constant region.

It will be apparent to the skilled person based on the disclosure hereinthat the present disclosure encompasses human, humanized, synhumanized,chimeric and primatized proteins.

The present disclosure also encompasses monoclonal antibodies. Forexample, the present disclosure provides an anti-Fn14 monoclonalantibody, the antibody comprising any one of the following:

-   (i) a V_(H) comprising a sequence set forth in SEQ ID NO: 13 and a    V_(L) comprising a sequence set forth in SEQ ID NO: 14;-   (ii) a V_(H) comprising a sequence set forth in SEQ ID NO: 15 and a    V_(L) comprising a sequence set forth in SEQ ID NO: 22;-   (iii) a V_(H) comprising a sequence set forth in SEQ ID NO: 16 and a    V_(L) comprising a sequence set forth in SEQ ID NO: 23;-   (iv) a V_(H) comprising a sequence set forth in SEQ ID NO: 17 and a    V_(L) comprising a sequence set forth in SEQ ID NO: 24;-   (v) a V_(H) comprising a sequence set forth in SEQ ID NO: 18 and a    V_(L) comprising a sequence set forth in SEQ ID NO: 25;-   (vi) a V_(H) comprising a sequence set forth in SEQ ID NO: 19 and a    V_(L) comprising a sequence set forth in SEQ ID NO: 26;-   (vii) a V_(H) comprising a sequence set forth in SEQ ID NO: 20 and a    V_(L) comprising a sequence set forth in SEQ ID NO: 27; or-   (viii) a V_(H) comprising a sequence set forth in SEQ ID NO: 21 and    a V_(L) comprising a sequence set forth in SEQ ID NO: 28.

In one example, an Fn14-binding protein or antibody of the presentdisclosure is conjugated to a compound. For example, the compound isselected from the group consisting of a radioisotope, a detectablelabel, a therapeutic compound, a colloid, a toxin, a nucleic acid, apeptide, a protein, a compound that increases the half life of theFn14-binding protein in a subject and mixtures thereof.

The present disclosure also provides an isolated or recombinant nucleicacid encoding the Fn14-binding protein or antibody of the disclosure.

The present disclosure additionally provides an expression constructcomprising the nucleic acid of the disclosure operably linked to apromoter. Such an expression construct can be in a vector, e.g., aplasmid.

In examples of the disclosure directed to single polypeptideFn14-binding proteins, the expression construct may comprise a promoterlinked to a nucleic acid encoding that polypeptide chain.

In examples directed to multiple polypeptides that form an Fn14-bindingprotein, an expression construct of the disclosure comprises a nucleicacid encoding one of the polypeptides (e.g., comprising a V_(H))operably linked to a promoter and a nucleic acid encoding another of thepolypeptides (e.g., comprising a V_(L)) operably linked to anotherpromoter.

In another example, the expression construct is a bicistronic expressionconstruct, e.g., comprising the following operably linked components in5′ to 3′ order:

-   (i) a promoter-   (ii) a nucleic acid encoding a first polypeptide;-   (iii) an internal ribosome entry site; and-   (iv) a nucleic acid encoding a second polypeptide.

For example, the first polypeptide comprises a V_(H) and the secondpolypeptide comprises a V_(L), or the first polypeptide comprises aV_(L) and the second polypeptide comprises a V_(H).

The present disclosure also contemplates separate expression constructsone of which encodes a first polypeptide (e.g., comprising a V_(H)) andanother of which encodes a second polypeptide (e.g., comprising aV_(L)). For example, the present disclosure also provides a compositioncomprising:

-   (i) a first expression construct comprising a nucleic acid encoding    a polypeptide (e.g., comprising a V_(H) operably linked to a    promoter); and-   (ii) a second expression construct comprising a nucleic acid    encoding a polypeptide

(e.g., comprising a V_(L) operably linked to a promoter),

wherein the first and second polypeptides associate to form anFn14-binding protein of the present disclosure.

The present disclosure additionally provides an isolated cell expressingthe Fn14-binding protein or antibody of the present disclosure or arecombinant cell genetically-modified to express an Fn14-binding proteinor antibody of the disclosure. In one example, the cell is an isolatedhybridoma. In another example, the cell comprises the nucleic acid of orthe expression construct of the disclosure or:

(i) a first expression construct comprising a nucleic acid encoding apolypeptide (e.g., comprising a V_(H)) operably linked to a promoter;and

(ii) a second expression construct comprising a nucleic acid encoding apolypeptide (e.g., comprising a V_(L)) operably linked to a promoter,

wherein the first and second polypeptides associate to form anFn14-binding protein or antibody of the present disclosure

The present disclosure additionally provides a composition comprisingthe Fn14-binding protein or the antibody or the nucleic acid or theexpression construct or the cell of the present disclosure and asuitable carrier. In one example, the composition comprises theFn14-binding protein or the antibody of the present disclosure.

In one example, the carrier is pharmaceutically acceptable.

The present disclosure additionally provides a method for treating orpreventing an Fn14-mediated condition in a subject, the methodcomprising administering the Fn14-binding protein or the antibody or thenucleic acid or the expression construct or the cell or the compositionof the present disclosure to the subject.

In one example, the Fn14-mediated condition is cancer, metastasis,tissue invasion by a cancer, excessive vascularization or angiogenesis,an autoimmune disease, an inflammatory disease, a neurodegenerativediseases, a wasting disorder, keloid scarring, graft versus hostdisease, graft rejection or ischemia.

In one example, the Fn14-mediated condition is an inflammatory diseaseor an autoimmune disease. In one example, the condition is a connectivetissue disease (including inflammatory arthritis, such as rheumatoidarthritis, psoriatic arthritis, reactive arthritis or gout), lupus(including systemic lupus erythematosus), type 1 diabetes, multiplesclerosis, vasculitis (including Wegener's granulomatosis and HenochSchonlein Syndrome), nephritis (including glomerulonephritis andpneumonitis), atherosclerosis or inflammation of the eye (includinguveitis).

In one example, the condition is cardiovascular disease.

In one example, the Fn14-mediated condition is selected from graftversus host disease, cardiac allograft vasculopathy, intramyocardialinfarction, ischemic reperfusion injury, connective tissue disease (suchas rheumatoid arthritis) or scleroderma.

As exemplified herein, the inventors have demonstrated that anFn14-binding protein disclosed herein is useful for reducing orpreventing invasiveness of a cancer into tissue of a subject. Suchinvasiveness is a component of cancer progression or metastasis.Accordingly, in one example, the Fn14-mediated condition is invasivenessof a cancer into tissue.

In one example, the Fn14-binding protein binds to recombinant human Fn14with a K_(D) of 1 nM or less, such as, 0.9 nM or less, for example, 0.8nM or less, for example, 0.7 nM or less, for example, 0.6 nM or less. Inone example, the K_(D) is assessed by immobilizing the Fn14-bindingprotein (e.g., a Fab or an antibody) and assessing binding of humanrecombinant Fn14 to the immobilized Fn14-binding protein using surfaceplasmon resonance.

In one example, the K_(D) is between about 0.01 nM and 1 nM, such asbetween about 0.05 nM and 0.9 nM, for example, between about 0.09 nM and0.7 nM, for example, between about 0.1 nM and 0.6 nM.

An exemplary Fn14-binding protein of the disclosure has a K_(D) of about0.5 nM (e.g., +/−0.1 nM) for recombinant human Fn14. For example, theFn14-binding protein is CRCBT-06-001 or an antibody comprising theantigen binding domain, variable regions or CDRs thereof.

Another exemplary Fn14-binding protein of the disclosure has a K_(D) ofabout 0.2 nM (e.g., +/−0.1 nM) for recombinant human Fn14. For example,the Fn-14 binding protein is CRCBT-06-002 or an antibody comprising theantigen binding domain, variable regions or CDRs thereof.

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising a proline or alanine substituted for thearginine at position 56 of SEQ ID NO: 1 at a similar or substantiallythe same level as it binds to an extracellular domain of Fn14 (e.g.,comprising a sequence set forth in SEQ ID NO: 2). For example, theFn14-binding protein binds to a peptide consisting of the sequence setforth in SEQ ID NO: 52 or 54 optionally additionally comprising sixhistidine residues at a terminus at a similar or substantially the samelevel as it binds to an extracellular domain of Fn14 (e.g., comprising asequence set forth in SEQ ID NO: 2). In one example, the level ofbinding is assessed by immobilizing the peptide and contacting thepeptide with the Fn14-binding protein. Exemplary Fn14-binding proteinsdescribed herein having such binding characteristics comprise thevariable regions and/or CDRs of an antibody designated CRCBT-06-001,CRCBT-06-002 or CRCBT-06-005. Thus, in one example, the Fn14-bindingprotein binds to a peptide consisting of the sequence set forth in SEQID NO: 52 or 54 (optionally with an additional six histidine residues ata terminus) at a similar or substantially the same level or with asimilar or substantially the same affinity as an antibody designatedCRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005. In another example, theFn14-binding protein competitively inhibits binding of an antibodydesignated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005 to a peptideconsisting of the sequence set forth in SEQ ID NO: 52 or 54 (optionallywith an additional six histidine residues at a terminus).

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising an alanine substituted for the arginine atposition 58 of SEQ ID NO: 1 at a similar or substantially the same levelas it binds to an extracellular domain of Fn14 (e.g., comprising asequence set forth in SEQ ID NO: 2). For example, the Fn14-bindingprotein binds to a peptide consisting of the sequence set forth in SEQID NO: 56 optionally additionally comprising six histidine residues at aterminus at a similar or substantially the same level as it binds to anextracellular domain of Fn14 (e.g., comprising a sequence set forth inSEQ ID NO: 2). In one example, the level of binding is assessed byimmobilizing the peptide and contacting the peptide with theFn14-binding protein or by expressing the peptide on the surface of aphage and contacting the phage to an immobilized Fn14 binding protein.Exemplary Fn14-binding proteins described herein having such bindingcharacteristics comprise the variable regions and/or CDRs of an antibodydesignated CRCBT-06-001, CRCBT-06-002 or CRCBT-06-005. Thus, in oneexample, the Fn14-binding protein binds to a peptide consisting of thesequence set forth in SEQ ID NO: 56 (optionally with an additional sixhistidine residues at a terminus) at a similar or substantially the samelevel or with a similar or substantially the same affinity as anantibody designated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005. Inanother example, the Fn14-binding protein competitively inhibits bindingof an antibody designated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005to a peptide consisting of the sequence set forth in SEQ ID NO: 56(optionally with an additional six histidine residues at a terminus).

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising an alanine substituted for the histidine atposition 60 of SEQ ID NO: 1 at a similar or substantially the same levelas it binds to an extracellular domain of Fn14 (e.g., comprising asequence set forth in SEQ ID NO: 2). For example, the Fn14-bindingprotein binds to a peptide consisting of the sequence set forth in SEQID NO: 63 optionally additionally comprising six histidine residues at aterminus at a similar or substantially the same level as it binds to anextracellular domain of Fn14 (e.g., comprising a sequence set forth inSEQ ID NO: 2). In one example, the level of binding is assessed byimmobilizing the peptide and contacting the peptide with theFn14-binding protein. Exemplary Fn14-binding proteins described hereinhaving such binding characteristics comprise the variable regions and/orCDRs of an antibody designated CRCBT-06-001 or CRCBT-06-002. Thus, inone example, the Fn14-binding protein binds to a peptide consisting ofthe sequence set forth in SEQ ID NO: 63 (optionally with an additionalsix histidine residues at a terminus) at a similar or substantially thesame level or with a similar or substantially the same affinity as anantibody designated CRCBT-06-001 or CRCBT-06-002. In another example,the Fn14-binding protein competitively inhibits binding of an antibodydesignated CRCBT-06-001 or CRCBT-06-002 to a peptide consisting of thesequence set forth in SEQ ID NO: 63 (optionally with an additional sixhistidine residues at a terminus).

In one example, the method comprises administering an antibodycomprising one or more of the following:

-   (i) a V_(H) comprising a sequence set forth in SEQ ID NO: 15 or a    humanized or deimmunized version thereof and a V_(L) comprising a    sequence set forth in SEQ ID NO: 22 or a humanized or deimmunized    version thereof or-   (ii) a V_(H) comprising a sequence set forth in SEQ ID NO: 16 or a    humanized or deimmunized version thereof and a V_(L) comprising a    sequence set forth in SEQ ID NO: 23 or a humanized or deimmunized    version thereof.

In one example, the method reduces invasiveness of a tumor into a tissuesurrounding the tumor. In one example, the method reduces invasivenessof a tumor into skeletal muscle.

Given the ability of the Fn14-binding protein to reduce tissue invasion,it is also useful for reducing or preventing tumor metastasis. Thus, thepresent disclosure additionally provides a method for preventingmetastasis or reducing the risk of metastasis in a subject sufferingfrom cancer, the method comprising administering the Fn14-bindingprotein or the antibody or the nucleic acid or the expression constructor the cell or the composition of the present disclosure to the subject.

As exemplified herein, the inventors have demonstrated that anFn14-binding protein disclosed herein is useful for treating a cancer(e.g., colon cancer) in a subject.

In one example, the Fn14-binding protein binds to recombinant human Fn14with a K_(D) of 0.4 nm, such as, 0.3 nM or less, for example, 0.25 nM orless. In one example, the K_(D) is assessed by immobilizing theFn14-binding protein (e.g., a Fab or an antibody) and assessing bindingof human recombinant Fn14 to the immobilized Fn14-binding protein usingsurface plasmon resonance.

In one example, the K_(D) is between about 0.01 nM and 0.3 nM, such asbetween about 0.05 nM and 0.25 nM.

Another exemplary Fn14-binding protein of the disclosure has a K_(D) ofabout 0.2 nM (e.g., +/−0.1 nM) for recombinant human Fn14. For example,the Fn-14 binding protein is CRCBT-06-002 or an antibody comprising theantigen binding domain, variable regions or CDRs thereof.

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising a proline or alanine substituted for thearginine at position 56 of SEQ ID NO: 1 at a similar or substantiallythe same level as it binds to an extracellular domain of Fn14 (e.g.,comprising a sequence set forth in SEQ ID NO: 2). For example, theFn14-binding protein binds to a peptide consisting of the sequence setforth in SEQ ID NO: 52 or 54 optionally additionally comprising sixhistidine residues at a terminus at a similar or substantially the samelevel as it binds to an extracellular domain of Fn14 (e.g., comprising asequence set forth in SEQ ID NO: 2). In one example, the level ofbinding is assessed by immobilizing the peptide and contacting thepeptide with the Fn14-binding protein. Exemplary Fn14-binding proteinsdescribed herein having such binding characteristics comprise thevariable regions and/or CDRs of an antibody designated CRCBT-06-001,CRCBT-06-002 or CRCBT-06-005. Thus, in one example, the Fn14-bindingprotein binds to a peptide consisting of the sequence set forth in SEQID NO: 52 or 54 (optionally with an additional six histidine residues ata terminus) at a similar or substantially the same level or with asimilar or substantially the same affinity as an antibody designatedCRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005. In another example, theFn14-binding protein competitively inhibits binding of an antibodydesignated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005 to a peptideconsisting of the sequence set forth in SEQ ID NO: 52 or 54 (optionallywith an additional six histidine residues at a terminus).

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising an alanine substituted for the arginine atposition 58 of SEQ ID NO: 1 at a similar or substantially the same levelas it binds to an extracellular domain of Fn14 (e.g., comprising asequence set forth in SEQ ID NO: 2). For example, the Fn14-bindingprotein binds to a peptide consisting of the sequence set forth in SEQID NO: 56 optionally additionally comprising six histidine residues at aterminus at a similar or substantially the same level as it binds to anextracellular domain of Fn14 (e.g., comprising a sequence set forth inSEQ ID NO: 2). In one example, the level of binding is assessed byimmobilizing the peptide and contacting the peptide with theFn14-binding protein or by expressing the peptide on the surface of aphage and contacting the phage to an immobilized Fn14 binding protein.Exemplary Fn14-binding proteins described herein having such bindingcharacteristics comprise the variable regions and/or CDRs of an antibodydesignated CRCBT-06-001, CRCBT-06-002 or CRCBT-06-005. Thus, in oneexample, the Fn14-binding protein binds to a peptide consisting of thesequence set forth in SEQ ID NO: 56 (optionally with an additional sixhistidine residues at a terminus) at a similar or substantially the samelevel or with a similar or substantially the same affinity as anantibody designated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005. Inanother example, the Fn14-binding protein competitively inhibits bindingof an antibody designated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005to a peptide consisting of the sequence set forth in SEQ ID NO: 56(optionally with an additional six histidine residues at a terminus).

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising an alanine substituted for the histidine atposition 60 of SEQ ID NO: 1 at a similar or substantially the same levelas it binds to an extracellular domain of Fn14 (e.g., comprising asequence set forth in SEQ ID NO: 2). For example, the Fn14-bindingprotein binds to a peptide consisting of the sequence set forth in SEQID NO: 63 optionally additionally comprising six histidine residues at aterminus at a similar or substantially the same level as it binds to anextracellular domain of Fn14 (e.g., comprising a sequence set forth inSEQ ID NO: 2). In one example, the level of binding is assessed byimmobilizing the peptide and contacting the peptide with theFn14-binding protein. Exemplary Fn14-binding proteins described hereinhaving such binding characteristics comprise the variable regions and/orCDRs of an antibody designated CRCBT-06-001 or CRCBT-06-002. Thus, inone example, the Fn14-binding protein binds to a peptide consisting ofthe sequence set forth in SEQ ID NO: 63 (optionally with an additionalsix histidine residues at a terminus) at a similar or substantially thesame level or with a similar or substantially the same affinity as anantibody designated CRCBT-06-001 or CRCBT-06-002. In another example,the Fn14-binding protein competitively inhibits binding of an antibodydesignated CRCBT-06-001 or CRCBT-06-002 to a peptide consisting of thesequence set forth in SEQ ID NO: 63 (optionally with an additional sixhistidine residues at a terminus).

In one example, the method comprises administering an antibodycomprising one or more of the following:

-   (i) a V_(H) comprising a sequence set forth in SEQ ID NO: 15 or a    humanized or deimmunized version thereof and a V_(L) comprising a    sequence set forth in SEQ ID NO: 22 or a humanized or deimmunized    version thereof; or-   (ii) a V_(H) comprising a sequence set forth in SEQ ID NO: 16 or a    humanized or deimmunized version thereof and a V_(L) comprising a    sequence set forth in SEQ ID NO: 23 or a humanized or deimmunized    version thereof.

The present disclosure additionally provides the Fn14-binding protein orthe antibody or the nucleic acid or the expression construct or the cellor the composition of the present disclosure for use in medicine.

The present disclosure additionally provides the Fn14-binding protein orthe antibody or the nucleic acid or the expression construct or the cellor the composition of the present disclosure for use in the treatment orprophylaxis of an Fn14-mediated condition. In one example, theFn14-mediated condition is cancer metastasis or tumor invasiveness.

The present disclosure additionally provides for use of the Fn14-bindingprotein or the antibody or the nucleic acid or the expression constructor the cell or the composition of the present disclosure in medicine.

The present disclosure additionally provides for use of the Fn14-bindingprotein or the antibody or the nucleic acid or the expression constructor the cell of the present disclosure in the manufacture of a medicamentfor the treatment or prophylaxis of an Fn14-mediated condition. In oneexample, the Fn14-mediated condition is cancer metastasis or tumorinvasiveness.

The present disclosure additionally provides a method for detecting Fn14in a sample, the method comprising contacting a sample with theFn14-binding protein or antibody of the disclosure such that anantigen-protein complex forms and detecting the complex, whereindetecting the complex is indicative of Fn14 in the sample. In oneexample, the sample is from a subject suffering from a Fn14-mediatedcondition.

The present disclosure additionally provides a method for diagnosing anFn14-mediated condition in a subject, the method comprising performingthe method described herein for detecting Fn14 in a sample from thesubject, wherein detection of Fn14 in the sample is indicative of thecondition.

In one example, the method comprises determining the level of Fn14 inthe sample, wherein an increased or decreased level of Fn14 in thesample compared to a control sample is indicative of the condition.

The present disclosure additionally provides a method for localizingand/or detecting and/or diagnosing and/or prognosing an Fn14-mediatedcondition, the method comprising detecting in vivo the Fn14-bindingprotein or antibody of the present disclosure bound to Fn14, if present,wherein the Fn14-binding protein or antibody is conjugated to adetectable tag.

In one example, the method additionally comprises administering thebinding molecule to the subject.

In one example of any method oftreatment/prophylaxis/diagnosis/prognosis described herein theFn14-mediated condition is cancer, metastasis, excessive vascularizationor angiogenesis, an autoimmune disease, an inflammatory disease, aneurodegenerative diseases, a wasting disorder or ischemia.

As discussed herein, the present inventors have also determined thatthey can treat or prevent a wasting disorder associated with a conditionusing an Fn14-binding protein that binds to Fn14. Accordingly, thepresent disclosure additionally provides a method of treating orpreventing a wasting disorder which is associated with a condition, themethod comprising administering to a subject an Fn14-binding proteincomprising an antigen binding domain of an anti-Fn14 antibody.

In one example, the wasting disorder is selected from the groupconsisting of unintended body weight loss, cachexia, pre-cachexia,muscle wasting and fat wasting. For example, the wasting disorder iscachexia.

In one example, the wasting disorder is associated with a conditionselected from the group consisting of cancer, metabolic acidosis,infectious diseases, diabetes, autoimmune immune deficiency syndrome(AIDS), autoimmune disorders, addiction to drugs, cirrhosis of theliver, chronic inflammatory disorders, anorexia, chronic heart failure,chronic kidney disease, osteoporosis, skeletal muscle disease, motorneuron disease, multiple sclerosis, muscle atrophy and neurodegenerativedisease.

In one example, the wasting disorder is cachexia, pre-cachexia orsarcopenia (e.g., muscle wasting associated with aging).

In one example, the wasting disorder is cachexia.

In one example, the cachexia is associated with cancer, infectiousdisease (e.g., tuberculosis or leprosy), AIDS, autoimmune disease(including rheumatoid arthritis or type 1 diabetes), cystic fibrosis,drug addiction, alcoholism or liver cirrhosis.

In one example, the cachexia is associated with a condition selectedfrom rheumatoid arthritis, diabetes, cardiac disease, chronic kidneydisease, chronic pulmonary inflammation, intestinal inflammation,inflammatory bowel disease, age, sepsis or AIDS.

In one exemplary form of the present disclosure the wasting disorder iscachexia associated with cancer. Numerous types of cancer are associatedwith cachexia, including solid tumors, carcinoma, neuroma, melanoma,leukemia, lymphoma, sarcoma, fibroma, thyroid cancer, bladder cancer,lung cancer, blastoma, bone cancer, bone tumor, brain stem glioma, braintumor, breast cancer, bronchial tumor, cervical cancer, colon cancer,colorectal cancer, neuroepithelial tumor, endometrial cancer,endometrial uterine cancer, fallopian tube cancer, kidney cancer, oralcancer, myeloma, neoplasm, neurinoma, neuroblastoma, ovarian cancer,pancreatic cancer, prostate cancer, rectal cancer or renal cellcarcinoma. Additional cancers are described herein.

In one exemplary form of the present disclosure the wasting disorder iscachexia associated with diabetes.

As discussed herein, the inventors have also determined that they cantreat a wasting disorder, e.g., muscle wasting (such as cachexia) bycontacting Fn14 on a tissue affected by the condition associated withthe wasting disorder. For example, the inventors have found that bycontacting Fn14 on a cancer they can treat muscle wasting. Thus, in someexamples, the method of the disclosure comprises administering an amountof the Fn14-binding protein effective to bind to Fn14 in a tissueaffected by the condition associated with the wasting disease, whereinthe tissue is not muscle tissue. In the case of cachexia associated withcancer, the method comprises administering an amount of the Fn14-bindingprotein effective to bind to Fn14 on the cancer cells.

In some examples, the method of the disclosure comprises administeringan Fn14-binding protein for a time and under conditions effective tobind to Fn14 in a tissue affected by the condition associated with thewasting disease, wherein the tissue is not muscle tissue. In the case ofcachexia associated with cancer, the method comprises administering anFn14-binding protein for a time and under conditions effective to bindto Fn14 on the cancer cells.

In one example, the method comprises administering the Fn14-bindingprotein to a subject suffering from a condition associated with awasting disorder, wherein the condition is associated with or caused bya cell expressing Fn14. For example, the method comprises administeringthe Fn14-binding protein to a subject suffering from cancer cachexia,wherein the subject suffers from a cancer expressing Fn14.

In one example, the method additionally comprises selecting a subjectsuffering from a wasting disorder associated with a condition associatedwith or caused by a cell expressing Fn14. For example, the methodadditionally comprises selecting a subject suffering from cancercachexia, wherein the subject suffers from a cancer expressing Fn14.

In one example, the Fn14-binding protein modulates Tweak-mediated Fn14signaling.

For example, the inventors have shown that antibodies that antagonizeTweak-mediated NFκB signaling are useful in the methods of thedisclosure. In one example, the Fn14-binding protein is the Fn14-bindingprotein or antibody of the present disclosure.

The inventors have also shown that antibodies that antagonize or agonizeTweak-mediated IL-8 secretion are useful in the methods of thedisclosure. The inventors have shown antibodies having a slow off-rateor high affinity dissociation constant are useful in the methods of thedisclosure. Such antibodies are shown herein to provide a superioreffect compared to other anti-Fn4 antibodies.

For example, the Fn14-binding protein binds to recombinant human Fn14with a K_(D) of 1 nM or less, such as, 0.9 nM or less, for example, 0.8nM or less, for example, 0.7 nM or less, for example, 0.6 nM or less. Inone example, the K_(D) is assessed by immobilizing the Fn14-bindingprotein (e.g., a Fab or an antibody) and assessing binding of humanrecombinant Fn14 to the immobilized Fn14-binding protein using surfaceplasmon resonance. Additional exemplary K_(D) values are describedherein and are to be taken to apply mutatis mutandis to this example ofthe disclosure.

In another example, the Fn14-binding protein binds to recombinant humanFn14 with a Kd of about 8×10⁻⁴s⁻¹ or less, such as, 5×10⁻⁴ s⁻¹ or less,for example, 4×10⁻⁴ s⁻¹ or less, for example, 2×10⁻⁴ s⁻¹ or less. In oneexample, the Kd is assessed by immobilizing the Fn14-binding protein(e.g., a Fab or an antibody) and assessing binding of recombinant humanFn14 to the immobilized Fn14-binding protein using surface plasmonresonance. Additional exemplary Kd values are described herein and areto be taken to apply mutatis mutandis to this example of the disclosure.

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising a proline or alanine substituted for thearginine at position 56 of SEQ ID NO: 1 at a similar or substantiallythe same level as it binds to an extracellular domain of Fn14 (e.g.,comprising a sequence set forth in SEQ ID NO: 2). For example, theFn14-binding protein binds to a peptide consisting of the sequence setforth in SEQ ID NO: 52 or 54 optionally additionally comprising sixhistidine residues at a terminus at a similar or substantially the samelevel as it binds to an extracellular domain of Fn14 (e.g., comprising asequence set forth in SEQ ID NO: 2). In one example, the level ofbinding is assessed by immobilizing the peptide and contacting thepeptide with the Fn14-binding protein. Exemplary Fn14-binding proteinsdescribed herein having such binding characteristics comprise thevariable regions and/or CDRs of an antibody designated CRCBT-06-001,CRCBT-06-002 or CRCBT-06-005. Thus, in one example, the Fn14-bindingprotein binds to a peptide consisting of the sequence set forth in SEQID NO: 52 or 54 (optionally with an additional six histidine residues ata terminus) at a similar or substantially the same level or with asimilar or substantially the same affinity as an antibody designatedCRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005. In another example, theFn14-binding protein competitively inhibits binding of an antibodydesignated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005 to a peptideconsisting of the sequence set forth in SEQ ID NO: 52 or 54 (optionallywith an additional six histidine residues at a terminus).

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising an alanine substituted for the arginine atposition 58 of SEQ ID NO: 1 at a similar or substantially the same levelas it binds to an extracellular domain of Fn14 (e.g., comprising asequence set forth in SEQ ID NO: 2). For example, the Fn14-bindingprotein binds to a peptide consisting of the sequence set forth in SEQID NO: 56 optionally additionally comprising six histidine residues at aterminus at a similar or substantially the same level as it binds to anextracellular domain of Fn14 (e.g., comprising a sequence set forth inSEQ ID NO: 2). In one example, the level of binding is assessed byimmobilizing the peptide and contacting the peptide with theFn14-binding protein or by expressing the peptide on the surface of aphage and contacting the phage to an immobilized Fn14 binding protein.Exemplary Fn14-binding proteins described herein having such bindingcharacteristics comprise the variable regions and/or CDRs of an antibodydesignated CRCBT-06-001, CRCBT-06-002 or CRCBT-06-005. Thus, in oneexample, the Fn14-binding protein binds to a peptide consisting of thesequence set forth in SEQ ID NO: 56 (optionally with an additional sixhistidine residues at a terminus) at a similar or substantially the samelevel or with a similar or substantially the same affinity as anantibody designated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005. Inanother example, the Fn14-binding protein competitively inhibits bindingof an antibody designated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005to a peptide consisting of the sequence set forth in SEQ ID NO: 56(optionally with an additional six histidine residues at a terminus).

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising an alanine substituted for the histidine atposition 60 of SEQ ID NO: 1 at a similar or substantially the same levelas it binds to an extracellular domain of Fn14 (e.g., comprising asequence set forth in SEQ ID NO: 2). For example, the Fn14-bindingprotein binds to a peptide consisting of the sequence set forth in SEQID NO: 63 optionally additionally comprising six histidine residues at aterminus at a similar or substantially the same level as it binds to anextracellular domain of Fn14 (e.g., comprising a sequence set forth inSEQ ID NO: 2). In one example, the level of binding is assessed byimmobilizing the peptide and contacting the peptide with theFn14-binding protein. Exemplary Fn14-binding proteins described hereinhaving such binding characteristics comprise the variable regions and/orCDRs of an antibody designated CRCBT-06-001 or CRCBT-06-002. Thus, inone example, the Fn14-binding protein binds to a peptide consisting ofthe sequence set forth in SEQ ID NO: 63 (optionally with an additionalsix histidine residues at a terminus) at a similar or substantially thesame level or with a similar or substantially the same affinity as anantibody designated CRCBT-06-001 or CRCBT-06-002. In another example,the Fn14-binding protein competitively inhibits binding of an antibodydesignated CRCBT-06-001 or CRCBT-06-002 to a peptide consisting of thesequence set forth in SEQ ID NO: 63 (optionally with an additional sixhistidine residues at a terminus).

In one example, the Fn14-binding protein is an antibody comprising oneor more of the following:

-   (i) a V_(H) comprising a sequence set forth in SEQ ID NO: 15 or a    humanized, synhumanized or deimmunized version thereof and a V_(L)    comprising a sequence set forth in SEQ ID NO: 22 or a humanized,    synhumanized or deimmunized version thereof; or-   (ii) a V_(H) comprising a sequence set forth in SEQ ID NO: 16 or a    humanized, synhumanized or deimmunized version thereof and a V_(L)    comprising a sequence set forth in SEQ ID NO: 23 or a humanized,    synhumanized or deimmunized version thereof.

In one example, the Fn14-binding protein is an antibody comprising aV_(H) comprising a sequence set forth in SEQ ID NO: 15 or a humanized,synhumanized or deimmunized version thereof and a V_(L) comprising asequence set forth in SEQ ID NO: 22 or a humanized, synhumanized ordeimmunized version thereof.

In one example, the Fn14-binding protein is an antibody comprising aV_(H) comprising a sequence set forth in SEQ ID NO: 16 or a humanized,synhumanized or deimmunized version thereof and a V_(L) comprising asequence set forth in SEQ ID NO: 23 or a humanized, synhumanized ordeimmunized version thereof.

In one example, the subject's body weight and/or general healthincreases within at least one week of administration of the Fn14-bindingprotein.

In one example, the subject's body weight and/or general healthincreases within at least one or two or three or four or five or sixdays of administration of the Fn14-binding protein.

In one example, the subject's body weight and/or general healthincreases within at least one or two or three or four or five or sixweeks of administration of the Fn14-binding protein.

In another example, the subject to who the Fn14-binding protein isadministered loses less body weight than a subject suffering from thewasting disorder to who the Fn14-binding protein is not administered.

In a further example, the subject's body weight remains detectablyincreased for at least 7 days after administration of the Fn14-bindingprotein.

In a still further example, the subject's body weight is not reduced toa level significantly below the weight prior to administration of theFn14-binding protein at least about 7 days after administration of theFn14-binding protein.

In another example, administration of the Fn14-binding protein extendsthe life of the subject compared to a subject or a population ofsubjects suffering from the wasting disorder to who the Fn14-bindingprotein has not been administered. In this regard, when comparing to apopulation of subjects, the extension of life may be compared to themean or the median term of life for the population.

In one example, administration of the Fn14-binding protein significantlyextends the life of the subject or a population of subjects to who it isadministered compared to a subject or a population of subjects sufferingfrom the wasting disorder to who the Fn14-binding protein has not beenadministered.

In one example, administration of the Fn14-binding protein extends thelife of the subject to who it is administered for a time sufficient topermit one or more additional treatments of the disorder associated withthe wasting compared to a subject or a population of subjects sufferingfrom the wasting disorder to who the Fn14-binding protein has not beenadministered.

In one example, the subject's life is extended by at least about 1 monthor 6 months or 24 months.

In one example, the subject's life is extended while the subject isreceiving treatment as described herein.

In another example, the subject's life is extended by 1% or 5% or 10% or20% or 40% of the term of life of a subject or a population of subjectssuffering from the wasting disorder to who the Fn14-binding protein hasnot been administered.

In one example, the Fn14-binding protein is administered at a dosebetween 0.5 mg/kg to about 20 mg/kg. For example, the Fn14-bindingprotein is administered at a dose between 1 mg/kg and 15 mg/kg, such asbetween 2 mg/kg and 10 mg/kg.

In one example, the Fn14-binding protein is administered at a dose of atleast about 2 mg/kg. For example, the Fn14-binding protein isadministered at a dose of at least about 3 mg/kg or 4 mg/kg or 5 mg/kgor 6 mg/kg or 7 mg/kg or 8 mg/kg or 9 mg/kg or 5 mg/kg. In one example,the Fn14-binding protein is administered at a dose between 2 mg/kg and20 mg/kg, such as, between 3 mg/kg and 15 mg/kg, for example, between 5mg/kg and 10 mg/kg, inclusive. In one example the Fn14-binding proteinis administered at a dose of about 5 mg/kg. In another example, theFn14-binding protein is administered at a dose of about 10 mg/kg.

In one example, the Fn14-binding protein is administered in multipledoses.

For example, the Fn14-binding protein is administered twice weekly forat least two weeks or three weeks or four weeks.

In another example, the Fn14-binding protein is administered in multipledoses each separated by at least about 7 days or 14 days or 21 days or28 days or one calendar month.

In a further example, the Fn14-binding protein is administered at aloading dose and at a maintenance dose. For example, the loading dose isbetween about 0.5 mg/kg and 2 mg/kg (such as between 0.5 mg/kg and 1mg/kg) and the maintenance dose of about 2 mg/kg to about 20 mg/kg (suchas between about 3 mg/kg and about 15 mg/kg, for example, between about5 mg/kg and 10 mg/kg, inclusive). In one example, the loading dose is adose of about 0.5 mg/kg or 1 mg/kg and the maintenance dose is about 5mg/kg or 10 mg/kg. This dosing regime is also called a stepped-up dosingregime.

In one example, the maintenance dose is administered about 5 days or 6days or 7 days or 8 days after administration of the loading dose. Forexample, the maintenance dose is administered about 7 days afteradministration of the loading dose

In one example, the method of treatment further comprises measuring thesubject's body weight prior to administering the Fn14-binding protein,and administering the Fn14-binding protein if the subject's weight hasdeclined by greater than about 5% within about 30 days.

In another example, the method further comprises measuring the subject'sbody weight prior to administering a dose of the Fn14-binding protein,and administering the Fn14-binding protein if the subject's weight hasdeclined by greater than approximately 1% since administration of aprevious dose of the Fn14-binding protein.

In a still further example, the method further comprises assessing thesubject's cachexia. For example, the subject's cachexia is assessed bymeasuring the subject's total body mass, lean body mass and/or body massindex. In one example, measurement of the subject's total body mass,lean body mass and/or body mass index does not include the estimatedweight of the subject's tumor(s) and/or extravascular fluidcollection(s).

In one example, the method additionally comprises treating the disorderassociated with the wasting disorder.

In one example, the method additionally comprises treating cancer, e.g.,a cancer associated with cachexia. For example, the treatment comprisesadministration of an anti-cancer drug or radiation therapy.

In one example, treatment for the cancer or disorder associated with thewasting disorder is performed or administered at the same time or afteradministering the Fn14-binding protein. For example, the Fn14-bindingprotein is administered at least once and the subject's weight permittedto increase prior to performing or administering the treatment forcancer or disorder associated with the wasting disorder.

The present disclosure also provides an Fn14-binding protein comprisingan antigen binding domain of an anti-Fn14 antibody for use in treatingor preventing a wasting disorder which is associated with a condition.

The present disclosure also provides for use of an Fn14-binding proteincomprising an antigen binding domain of an anti-Fn14 antibody in themanufacture of a medicament for treating or preventing a wastingdisorder which is associated with a condition.

The present inventors have shown that an Fn14-binding protein disclosedherein is useful for reducing or preventing invasiveness of a tumor intomuscle of a subject. Accordingly, one specific example of the presentdisclosure provides a method for reducing or preventing tumor invasioninto muscle of a subject suffering from cancer, the method comprisingadministering to the subject an Fn14-binding protein comprising anantigen binding domain of an anti-Fn14 antibody, wherein theFn14-binding protein neutralizes a Tweak activity in a cell expressingFn14.

In one example, the Fn14-binding protein reduces Tweak-inducedNFκB-signaling in a cell expressing Fn14.

In one example, the Fn14-binding protein reduces Tweak-inducedNFκB-signaling in a cell expressing Fn14, and wherein the Fn14-bindingprotein does not detectably induce NFκB-signaling when contacted to acell expressing Fn14 in the absence of Tweak.

In one example, the method comprises administering the Fn14-bindingprotein or antibody of the present disclosure. In one example, themethod comprises administering an antibody comprising one or more of thefollowing:

-   (i) a V_(H) comprising a sequence set forth in SEQ ID NO: 15 or a    humanized or deimmunized version thereof and a V_(L) comprising a    sequence set forth in SEQ ID NO: 22 or a humanized, synhumanized or    deimmunized version thereof; or-   (ii) a V_(H) comprising a sequence set forth in SEQ ID NO: 16 or a    humanized or deimmunized version thereof and a V_(L) comprising a    sequence set forth in SEQ ID NO: 23 or a humanized, synhumanized or    deimmunized version thereof.

In one example, the method reduces invasiveness of a tumor into a tissuesurrounding the tumor. In one example, the method reduces invasivenessof a tumor into skeletal muscle.

In one example, the Fn14-binding protein is administered at the sametime as or prior to treatment for the tumor. Such treatment is usefulfor reducing metastases that can be induced or associated with treatmentof tumor.

The present disclosure also provides an Fn14-binding protein comprisingan antigen binding domain of an anti-Fn14 antibody for use in treatingor preventing tumor invasion into a muscle.

The present disclosure also provides for use of an Fn14-binding proteincomprising an antigen binding domain of an anti-Fn14 antibody in themanufacture of a medicament for treating or preventing tumor invasioninto a muscle.

The inventors have also shown that antibodies that antagonizeTweak-mediated NFκB signaling are useful in treating or preventing aglucose-metabolism disorder, such as diabetes, e.g., Type 1 diabetes. Inone example, the Fn14-binding protein is the Fn14-binding protein orantibody of the present disclosure.

The inventors have also shown that antibodies that antagonize or agonizeTweak-mediated IL-8 secretion are useful in the methods of thedisclosure. The inventors have shown antibodies having a slow off-rateor high affinity dissociation constant are useful in the methods of thedisclosure. Such antibodies are shown herein to provide a superioreffect compared to other anti-Fn4 antibodies.

For example, the Fn14-binding protein binds to recombinant human Fn14with a K_(D) of 1 nM or less, such as, 0.9 nM or less, for example, 0.8nM or less, for example, 0.7 nM or less, for example, 0.6 nM or less, or0.3 nM or less or 0.25 nM or less. In one example, the K_(D) is assessedby immobilizing the Fn14-binding protein (e.g., a Fab or an antibody)and assessing binding of human recombinant Fn14 to the immobilizedFn14-binding protein using surface plasmon resonance. Additionalexemplary K_(D) values are described herein and are to be taken to applymutatis mutandis to this example of the disclosure.

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising a proline or alanine substituted for thearginine at position 56 of SEQ ID NO: 1 at a similar or substantiallythe same level as it binds to an extracellular domain of Fn14 (e.g.,comprising a sequence set forth in SEQ ID NO: 2). For example, theFn14-binding protein binds to a peptide consisting of the sequence setforth in SEQ ID NO: 52 or 54 optionally additionally comprising sixhistidine residues at a terminus at a similar or substantially the samelevel as it binds to an extracellular domain of Fn14 (e.g., comprising asequence set forth in SEQ ID NO: 2). In one example, the level ofbinding is assessed by immobilizing the peptide and contacting thepeptide with the Fn14-binding protein. Exemplary Fn14-binding proteinsdescribed herein having such binding characteristics comprise thevariable regions and/or CDRs of an antibody designated CRCBT-06-001,CRCBT-06-002 or CRCBT-06-005. Thus, in one example, the Fn14-bindingprotein binds to a peptide consisting of the sequence set forth in SEQID NO: 52 or 54 (optionally with an additional six histidine residues ata terminus) at a similar or substantially the same level or with asimilar or substantially the same affinity as an antibody designatedCRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005. In another example, theFn14-binding protein competitively inhibits binding of an antibodydesignated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005 to a peptideconsisting of the sequence set forth in SEQ ID NO: 52 or 54 (optionallywith an additional six histidine residues at a terminus).

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising an alanine substituted for the arginine atposition 58 of SEQ ID NO: 1 at a similar or substantially the same levelas it binds to an extracellular domain of Fn14 (e.g., comprising asequence set forth in SEQ ID NO: 2). For example, the Fn14-bindingprotein binds to a peptide consisting of the sequence set forth in SEQID NO: 56 optionally additionally comprising six histidine residues at aterminus at a similar or substantially the same level as it binds to anextracellular domain of Fn14 (e.g., comprising a sequence set forth inSEQ ID NO: 2). In one example, the level of binding is assessed byimmobilizing the peptide and contacting the peptide with theFn14-binding protein or by expressing the peptide on the surface of aphage and contacting the phage to an immobilized Fn14 binding protein.Exemplary Fn14-binding proteins described herein having such bindingcharacteristics comprise the variable regions and/or CDRs of an antibodydesignated CRCBT-06-001, CRCBT-06-002 or CRCBT-06-005. Thus, in oneexample, the Fn14-binding protein binds to a peptide consisting of thesequence set forth in SEQ ID NO: 56 (optionally with an additional sixhistidine residues at a terminus) at a similar or substantially the samelevel or with a similar or substantially the same affinity as anantibody designated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005. Inanother example, the Fn14-binding protein competitively inhibits bindingof an antibody designated CRCBT-06-001 or CRCBT-06-002 or CRCBT-06-005to a peptide consisting of the sequence set forth in SEQ ID NO: 56(optionally with an additional six histidine residues at a terminus).

In one example, the Fn14-binding protein binds to an extracellulardomain of Fn14 comprising an alanine substituted for the histidine atposition 60 of SEQ ID NO: 1 at a similar or substantially the same levelas it binds to an extracellular domain of Fn14 (e.g., comprising asequence set forth in SEQ ID NO: 2). For example, the Fn14-bindingprotein binds to a peptide consisting of the sequence set forth in SEQID NO: 63 optionally additionally comprising six histidine residues at aterminus at a similar or substantially the same level as it binds to anextracellular domain of Fn14 (e.g., comprising a sequence set forth inSEQ ID NO: 2). In one example, the level of binding is assessed byimmobilizing the peptide and contacting the peptide with theFn14-binding protein. Exemplary Fn14-binding proteins described hereinhaving such binding characteristics comprise the variable regions and/orCDRs of an antibody designated CRCBT-06-001 or CRCBT-06-002. Thus, inone example, the Fn14-binding protein binds to a peptide consisting ofthe sequence set forth in SEQ ID NO: 63 (optionally with an additionalsix histidine residues at a terminus) at a similar or substantially thesame level or with a similar or substantially the same affinity as anantibody designated CRCBT-06-001 or CRCBT-06-002. In another example,the Fn14-binding protein competitively inhibits binding of an antibodydesignated CRCBT-06-001 or CRCBT-06-002 to a peptide consisting of thesequence set forth in SEQ ID NO: 63 (optionally with an additional sixhistidine residues at a terminus).

In one example, the Fn14-binding protein is an antibody comprising oneor more of the following:

-   (i) a V_(H) comprising a sequence set forth in SEQ ID NO: 15 or a    humanized, synhumanized or deimmunized version thereof and a V_(L)    comprising a sequence set forth in SEQ ID NO: 22 or a humanized,    synhumanized or deimmunized version thereof; or-   (ii) a V_(H) comprising a sequence set forth in SEQ ID NO: 16 or a    humanized, synhumanized or deimmunized version thereof and a V_(L)    comprising a sequence set forth in SEQ ID NO: 23 or a humanized,    synhumanized or deimmunized version thereof.

In one example, the Fn14-binding protein is an antibody comprising aV_(H) comprising a sequence set forth in SEQ ID NO: 15 or a humanized,synhumanized or deimmunized version thereof and a V_(L) comprising asequence set forth in SEQ ID NO: 22 or a humanized, synhumanized ordeimmunized version thereof.

In one example, the Fn14-binding protein is an antibody comprising aV_(H) comprising a sequence set forth in SEQ ID NO: 16 or a humanized,synhumanized or deimmunized version thereof and a V_(L) comprising asequence set forth in SEQ ID NO: 23 or a humanized, synhumanized ordeimmunized version thereof.

The inventors have also produced a model of a wasting disorder, e.g.,cancer cachexia. Accordingly, the present disclosure also provides anon-human mammal comprising tumor cells, wherein the tumor cells capableof ectopically expressing recombinant human Fn14, and wherein thenon-human mammal develops a wasting disorder. The present disclosurealso provides the tumor cells capable of ectopically expressingrecombinant human Fn14.

The present disclosure also provides a method for producing a non-humananimal model of a wasting disorder, the method comprising administeringto a non-human mammal a tumor cell of expressing recombinant human Fn14under conditions sufficient for the Fn14 to be expressed and for thewasting disorder to develop.

In one example, the Fn14 is inducibly expressed. For example, Fn14encoding nucleic acid is operably linked to a promoter induced by4-hydroxytamoxifen.

In one example, the Fn14 is constitutively expressed.

In one example, the tumor cell is a fibroblast cell genetically modifiedto express v12Hras.

The inventors have also produced a non-signaling form of Fn14 useful asa control in experiments to assess the effect of a compound in a wastingcondition, e.g., cancer cachexia. Animals to which tumor cellsexpressing the non-signaling fusion protein has been administereddevelop cancer, but do not develop the wasting condition.

In one example, the present disclosure provides a fusion proteincomprising the extracellular region of Fn14 and aglycosyl-phosphatidylinositol anchor (GPI) region of a receptor butlacking the cytosolic region of the receptor. For example, the fusionprotein does not mediate Tweak signaling when expressed in a cell

The present disclosure also provides a recombinant cell expressing afusion protein comprising an extracellular region of Fn14 and aglycosylphosphatidylinositol anchor (GPI) region of a receptor butlacking the cytosolic region of the receptor, wherein the fusion proteindoes not mediate Tweak signaling in the cell.

In one example, the extracellular region of Fn14 is the extracellularregion of human Fn14.

In one example, the GPI region is from Trail Receptor 3.

For example, the fusion protein comprises a sequence set forth in SEQ IDNO: 36 or between residues 28 to 126 of SEQ ID NO: 36.

The present disclosure also provides a method of identifying a compoundfor the treatment of a wasting disorder, the method comprising:

-   (i) administering the compound to the non-human mammal model of a    wasting disorder described herein and inducing expression of Fn14    (if necessary) and determining the level of the wasting disorder;-   (ii) comparing the level of the wasting disorder at (i) to the    levels of a wasting disorder in a non-human mammal administered the    cell expressing a fusion protein comprising an extracellular region    of Fn14 and a glycosylphosphatidylinositol anchor (GPI) region of a    receptor but lacking the cytosolic region of the receptor and    determining the level of the wasting disorder,-   wherein a similar level of the wasting disorder at (i) compared    to (ii) indicates that the compound is useful for treating a wasting    disorder.

The present disclosure also provides a method of identifying a compoundfor the treatment of a wasting disorder, the method comprising:

-   (i) administering the compound to the non-human mammal model of a    wasting disorder described herein and inducing expression of Fn14    (if necessary) and determining the level of the wasting disorder;-   (ii) administering to a non-human mammal the cell expressing a    fusion protein comprising an extracellular region of Fn14 and a    glycosylphosphatidylinositol anchor (GPI) region and determining the    level of the wasting disorder,    wherein a similar level of the wasting disorder at (i) compared    to (ii) indicates that the compound is useful for treating a wasting    disorder.

In one example, the non-human mammal expressing a fusion proteincomprising an extracellular region of Fn14 and aglycosylphosphatidylinositol anchor (GPI) region and determining thelevel of the wasting disorder, is also administered the compound.

In one example, the method additionally comprises isolating or providingthe compound or the structure of the compound.

In one example, the compound is an Fn14-binding protein comprising anantigen binding domain of an anti-Fn14 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to B is a series of graphical representations showing resultsof screening of sera from mice immunized with a recombinant proteincomprising the extracellular domain of hFn14. Panel A shows results ofassays in which live MEFv12Hras cells expressing human Fn14 (+/−4-OHTinduction) were stained with the serum from immunized mice andsubsequently analyzed by flow cytometry. Histogram traces: dotted tracerepresents staining on un-induced cells and solid trace representstaining on induced cells. Panel A.i. shows the cell population gated onside and forward scatter for analysis of all samples. Panel Aii. showsunstained cells, Panels Aiii. & Aiv. show cells stained with sera from 2un-immunized mice, Panels Av. & Avi. show sera from 2 mice immunizedwith a non-related antigen in PBS or adjuvant, respectively. Panel Avii.show anti-hFn14 positive control. Panel B. shows results using serumfrom mice immunized with cells suspended in PBS or Adjuvant as indicated(mice #1318-1323), with dotted boxes indicating sera from micedisplaying a positive immune response to hFn14. All sera samples wereused at 1:50 dilution for staining.

FIGS. 2A to B is a series of graphical representations showing resultsof screening of sera from mice immunized with recombinant Fn14-Fc onhuman D645 cells. Panel A shows results of screening using live D645human glioma cells stained with mouse serum or controls and analyzed byflow cytometry. Dotted histogram traces represent unstained cells andsolid traces represent stained cells. Panel A.i. shows a cell populationgated for analysis of all samples, Panel ii. shows unstained cells,Panel iii. shows anti-hFn14 positive control. Panels iv. & v. show cellsstained with serum from 2 un-immunized mice. Panels vi. & vii. showserum from 2 mice immunized with a non-related antigen in adjuvant orPBS, respectively. Panel B. shows sera from mice immunized with arecombinant protein comprising the extracellular domain of hFn14 inadjuvant or PBS as indicated (mice#1318-1323), with dotted boxesindicating mice with positive immune response. All sera samples wereused at 1:50 dilution for staining.

FIGS. 3A to C is a series of graphical representations showing detectionof monoclonal antibodies that specifically recognize cell surfaceexpressed human Fn14. Human Fn14 inducible MEFv12Hras cells (+/−4-OHT asindicated) were stained with control antibodies (Panel A) or hybridomasupernatant for a selection of hybridomas (Panel B) or hybridomasupernatant from clones CRCBT-06-001, CRCBT-06-002, CRCBT-06-003 andCRCBT-06-004 (Panel C). Solid traces represent supernatant staining ofcells and dotted traces represent the overlay of a secondary antibodyalone staining as a control (from panel A).

FIGS. 4A to C is a series of graphical representations showing theeffect of anti-hFn14 positive monoclonal antibodies on NFκB activation.HEK293T cells harboring an NFκB-responsive promoter operably linked to aGFP reporter were incubated for 24 hours in the presence/absence ofTweak-Fc (concentrations as indicated) and either control antibodies(Panel A) or supernatants from parent hybridoma clones (as indicated,Panel B) or purified monoclonal antibody CRCBT-06-001 at varyingconcentrations (as indicated, Panel C). Cells were harvested and GFPfluorescence assessed by flow cytometry. All supernatants were diluted1:10 for this assay. For comparison the dotted trace overlay representscells alone or in the presence of Tweak-Fc only at each concentration.Spn/t; supernatant

FIGS. 5A to B is a series of graphical representations showing resultsfrom screening of sera from mice immunized with HEK293T cellsover-expressing hFn14. Live MEFv12Hras cells expressing human Fn14(+/−4-OHT induction) were mixed at a 1:1 ratio and stained using serumfrom controls (Panel A) or immunized mice (Panel B). Cells were thenanalyzed by flow cytometry. Dotted histogram traces represent stainingof cells with serum from a mouse immunized with cells expressing anon-related antigen for comparison. Panels A.i. & ii. show staining ofcells with serum from mice immunized with HEK293T cells expressing othernon-related human proteins, Panel Aiii. shows anti-hFn14 positivecontrol and Panel Aiv. shows cells stained with secondary antibodyalone. Panel B. Serum from mice immunized with HEK293T cells expressinghFn14 suspended in PBS or Adjuvant as indicated (mice#31-36), Positiveresponse assessed based on appearance of a double peak as indicated bydotted box.

FIG. 6A is a series of graphical representations showing the effect ofpurified CRCBT-06-001, CRCBT-06-002, CRCBT-06-003 and CRCBT-06-004 onTweak-induced NFκB activation. HEK293T cells harboring anNFκB-responsive promoter operably linked to a GFP reporter wereincubated for 24 hours in the presence/absence of Tweak-Fc(concentrations as indicated) and purified antibody CRCBT-06-001,CRCBT-06-002, CRCBT-06-003 or CRCBT-06-004. Cells were harvested and GFPfluorescence assessed by flow cytometry. All supernatants were diluted1:50 for this assay. For comparison, the dotted trace overlay representscells alone or in the presence of Tweak-Fc only (as depicted in FIG. 6Cpanel i).

FIG. 6B is a series of graphical representations showing the effect ofpurified negative control antibody on Tweak induced NFκB activation.HEK293T cells harboring an NFκB-responsive promoter operably linked to aGFP reporter were incubated for 24 hours in the presence/absence ofTweak-Fc (concentrations as indicated) and purified control antibody.Cells were harvested and GFP fluorescence assessed by flow cytometry.For comparison, the dotted trace overlay represents cells alone or inthe presence of Tweak-Fc only (as in FIG. 6C Panel i.).

FIG. 6C is a series of graphical representations showing the effect ofcontrols (as indicated) on Tweak induced NFκB activation. HEK293T cellsharboring an NFκB-responsive promoter operably linked to a GFP reporterwere incubated for 24 hours in the presence/absence of Tweak-Fc(concentrations as indicated) and controls. Cells were harvested and GFPfluorescence assessed by flow cytometry. All supernatants were diluted1:50 for this assay. For comparison, the dotted trace overlay representscells alone or in the presence of Tweak-Fc only (as in panel i.).

FIG. 7A is a series of graphical representations showing the effect ofpurified CRCBT-06-001, CRCBT-06-005, CRCBT-06-006, CRCBT-06-007, ITEM-1and ITEM-2 on Tweak-induced NFκB activation. HEK293T cells harboring anNFκB-responsive promoter operably linked to a GFP reporter wereincubated for 24 hours in the presence/absence of Tweak-Fc (100 ng/ml or200 ng/ml) and either cells alone, isotype control IgG2b (dashed trace),purified CRCBT-06-001, CRCBT-06-005, CRCBT-06-006 or CRCBT-06-007 (solidtraces; at the concentrations indicated). Cells were harvested and GFPfluorescence assessed by flow cytometry.

FIG. 7B is a series of graphical representations showing the effect ofpurified CRCBT-06-001, ITEM-1 and ITEM-2 on Tweak-induced NFκBactivation. HEK293T cells harboring an NFκB-responsive promoter operablylinked to a GFP reporter were incubated for 24 hours in thepresence/absence of Tweak-Fc (100 ng/ml or 200 ng/ml) and either cellsalone (dashed trace as underlay for antibody graphs), purifiedCRCBT-06-001, ITEM-1 or ITEM-2 (solid traces; at the concentrationsindicated). Cells were harvested and GFP fluorescence assessed by flowcytometry.

FIG. 8 is a graphical representation showing results of an assay todetermine the ability of purified CRCBT-06-001, CRCBT-06-002,CRCBT-06-003 and CRCBT-06-004 to block Tweak-induced Kym1 cell death.Kym1 cells were incubated for 24 hours in the presence/absence ofTweak-Fc (concentrations as indicated) and purified antibody (at theconcentrations indicated) or TweakR-Fc as positive control. Total cellswere harvested and incubated with propidium iodide. Cells were analyzedby flow cytometry and graphed as a percentage of cell death in eachsample.

FIG. 9 is a graphical representation showing results of IL-8 secretionassay for assessing agonistic properties of antibodies. A375 cells wereincubated in the presence of antibody (10, 1, 0.1 or 0.01 μg/ml) for 24hours. Cell culture supernatants were assessed for amount of IL-8.Experiments were conducted with 3 independent biological replicates andthe results averaged and graphed as IL-8 (pg/ml) with error barsrepresenting the SEM.

FIG. 10 is a graphical representation showing IL-8 secretion assay fordetermination of antibody antagonist properties of antibodies. A375cells were incubated in the presence or absence of antibody (10 μg/ml)in the presence or absence of Tweak-Fc (300 ng/ml) for 24 hours. Cellculture supernatants were assessed for amount of IL-8. Experiments wereconducted with 3 independent biological replicates and the resultsaveraged and graphed as IL-8 (pg/ml) with error bars representing theSEM.

FIG. 11A is a diagrammatic representation showing sequences of the lightchains variable regions of monoclonal antibodies CRCBT-06-001,CRCBT-06-002, CRCBT-06-003, CRCBT-06-004, CRCBT-06-005, CRCBT-06-006 andCRCBT-06-007 (labeled 001, 002, 003, 004, 005, 006 and 007,respectively). The sequences have been aligned. CDRs 1-3 and frameworkregions 1-4 are indicated. CDRs according to the Kabat numbering systemare indicated in bold text. CDRs according to the Chothia numberingsystem are indicated in underlined text.

FIG. 11B is a diagrammatic representation showing sequences of a classof highly related light chains variable regions of monoclonal antibodiesdepicted in FIG. 11A (labeled 001, 002, 00.3, 004, 005 and 006). Thesequences have been aligned. CDRs 1-3 and flanking regions 1-4 areindicated. CDRs according to the Kabat numbering system are indicated inbold text. CDRs according to the Chothia numbering system are indicatedin underlined text. A consensus sequence is also shown in which “X”indicates a site of variation. Beneath the “X” is indicated all aminoacids that occur at that site in the analyzed sequences.

FIG. 11C is a diagrammatic representation showing sequences of the heavychains variable regions of monoclonal antibodies CRCBT-06-001,CRCBT-06-002, CRCBT-06-003, CRCBT-06-004, CRCBT-06-005, CRCBT-06-006 andCRCBT-06-007 (labeled 001, 002, 003, 004, 005, 006 and 007,respectively). The sequences have been aligned. CDRs 1-3 and flankingregions 1-4 are indicated. CDRs according to the Kabat numbering systemare indicated in bold text. CDRs according to the Chothia numberingsystem are indicated in underlined text.

FIG. 11D is a diagrammatic representation showing sequences of a classof highly related heavy chains variable regions of monoclonal antibodiesdepicted in FIG. 11C (labeled 001, 002, 005, 006 and 007). The sequenceshave been aligned. CDRs 1-3 and flanking regions 1-4 (defined accordingto the Kabat numbering system) are indicated. CDRs according to theKabat numbering system are indicated in bold text. CDRs according to theChothia numbering system are indicated in underlined text. A consensussequence is also shown in which “X” indicates a site of variation.Beneath the “X” is indicated all amino acids that occur at that site inthe analyzed sequences.

FIG. 12A is a series of graphical representations showing anti-Fn14antibody CRCBT-06-001 recognizes a conformational epitope. Panel A.i.shows CRCBT-06-001 binds to native Fn14 but not to reduced and alkylated(R+A) Fn14 (squares) by ELISA. Panel Aii. shows Native Fn14 and R+A Fn14retain binding to anti-Fc antibody indicating the integrity of R+A Fn14is retained. ♦=native Fn14, ▪=R+A Fn14, ▴=Blank.

FIG. 12B is a copy of a photographic representation showing results ofWestern blot analysis of Fn14 and R+A Fn14 showing the R+A Fn14 is notdetected by CRCBT-06-001 whereas the native Fn14 is detected strongly.Lanes labeled 1 and 2 Fn14-Fc preparation 1: Lanes 1=native Fn14-Fc,lanes 2=R+A Fn14, Lanes 3 and 4=Fn14-Fc preparation 2: lanes 3=nativeFn14-Fc, lanes 4=R+A Fn14-Fc. M=molecular weight markers. α-Fc=detectionusing and anti-Fc antibody. αFn14=detection using CRCBT-06-001

FIG. 12C is a graphical representation showing CRCBT-06-001,CRCBT-06-002, CRCBT-06-003, CRCBT-06-004, CRCBT-06-005, CRCBT-06-006 andCRCBT-06-007 recognize a conformational epitope. Antibodies were assayedby ELISA for binding to native and reduced and alkylated Fn14-Fc.Antigen was coated on the plate at 2 μg/ml and antibodies assayed at 1μg/ml. Anti-human Fc recognized the Fc portion of Fn14 indicating thatthe integrity of R+A Fn14 is retained. An irrelevant antibody (5G8) andsecondary antibody alone (anti-mouse HRP) were used as negativecontrols.

FIG. 13 is a series of representations. Panel A shows sequences of aselection of clones tested for binding to CRCBT-06-001. The shortestfragment identified by panning the phage display library (RW129) ishighlighted in bold. Panel B is a graphical representation showingbinding of the fragments listed in Panel A to CRCBT-06-001.

FIG. 14A is a series of graphical representations showing hFn14extracellular domain constructs for display on phage. Panel A shows analignment of the hFn14 constructs displayed on phage including: Thefull-length extracellular domain of hFn14; The D45A, K48A, M50A and D62Emutants, all known to have reduced affinity for the natural ligandTweak; and Sub-domains 1-3.

FIG. 14B shows pymol images of the solution structure solved by NMR ofthe human Fn14 extracellular domain structure (adapted from He et al.,2009). Subdomains 1 and 2 (SD1 and SD2, respectively) are depicted asare residues K48, D45, M50 and D62, which are known to be important forTweak binding.

FIG. 15 is a series of graphical representations showing hFn14extracellular domain constructs bind differentially to recombinantTweak-Fc. Panel A shows results of an ELISA comparing the reactivity ofeach normalized hFn14 construct with MAb 9E10. Each phage preparationwas 10-fold serially diluted and added to MAb 9E10 attached to the solidphase. Panel B shows results of the same ELISA as in Panel A comparingthe binding of the highest concentration of phage with MAb 9E10 and MAb5G8. Panel C shows results of an ELISA comparing the reactivity of eachnormalized phage-displayed hFn14 construct with recombinant Tweak-Fc.Panel D shows results of the same ELISA as in Panel C comparing thebinding of the highest concentration of each phage preparation withrecombinant Tweak-Fc and recombinant Fc alone. Binding was detectedusing HRP-conjugated anti-M13-phage polyclonal IgG and TMB substrate.Optical density at 450 nm was quantified using a Spectramaxspectrophotometer. Error bars for each dilution represent the standarddeviation of duplicate assays. In this figure, reference to “FN14Ectodomain” or “WT” is a reference to the extracellular domain of hFn14.Methods according to Epitope mapping using phage expressed Fn14—Method1.

FIG. 16A is a series of graphical representations showingphage-displayed hFn14 constructs bind to anti-Fn14 MAbs. ELISAs showingthe reactivity of the repertoire of hFn14 phage constructs with theanti-Fn14 monoclonal antibodies CRCBT-06-001, CRCBT-06-002, CRCBT-06-003and CRCBT-06-004. Binding was detected using HRP-conjugatedanti-M13-phage polyclonal IgG and TMB substrate. Optical density at 450nm was quantified using a Spectramax spectrophotometer. Error bars foreach dilution represent the standard deviation of duplicate assays. Inthis figure, reference to “FN14 Ectodomain” is a reference to theextracellular domain of hFn14. Methods according to Epitope mappingusing phage expressed Fn14—Method 1.

FIG. 16B is a series of graphical representations showingphage-displayed hFn14 extracellular domain constructs bind specificallyto anti-Fn14 MAbs. Results depicted are from the same ELISAs as in FIG.16A, comparing the reactivity of the repertoire of hFn14 phageconstructs with the anti-Fn14 monoclonal antibodies CRCBT-06-001,CRCBT-06-002, CRCBT-06-003 and CRCBT-06-004 and MAb 5G8 (as indicated).Binding was detected using HRP-conjugated anti-M13-phage polyclonal IgGand TMB substrate. Optical density at 450 nm was quantified using aSpectramax spectrophotometer. Error bars for each dilution represent thestandard deviation of duplicate assays. In this figure, reference to“WT” is a reference to the extracellular domain of hFn14.

FIG. 17 is a series of graphical representations showing anti-Fn14monoclonal antibody ITEM-1 binds well to all 4 Tweak mutants. Panel Ashows results of ELISAs showing the reactivity of the repertoire ofhFn14 phage constructs with MAb 9E10 and the anti-Fn14 monoclonalantibodies CRCBT-06-001 and ITEM-1. The concentration of each phagepreparation was adjusted to give an approximately equal reactivity withMAb 9E10, to normalize the amount of displayed hFn14 in each phagepreparation. Each normalized preparation was 10-fold serially dilutedand added in solution to the MAbs attached to the solid phase. AllELISAs shown here were performed simultaneously with the samepreparations of phage. Binding was detected using HRP-conjugatedanti-M13 phage polyclonal IgG and TMB substrate. Optical density at 450nm was quantified using a Spectramax spectrophotometer. Error bars foreach dilution represent the standard deviation of duplicate assays.Panel B shows the results of the same ELISAs as in Panel A showing thatnone of the phage-displayed hFn14 constructs bind significantly to thenegative control MAb 5G8. In this figure, reference to “FN14” is areference to the extracellular domain of hFn14. Methods according toEpitope mapping using phage expressed Fn14—Method 1.

FIG. 18A is a series of graphical representations showing anti-Fn14monoclonal antibodies bind specifically to sub-domain 2 of the hFn14extracellular domain. Results of ELISAs are depicted showing thereactivity of the phage-displayed hFn14 extracellular domain, sub-domain1 (SD1) and sub-domain 2 (SD2) with the anti-Fn14 monoclonal antibodiesCRCBT-06-001, CRCBT-06-002, CRCBT-06-003, CRCBT-06-004, CRCBT-06-005,CRCBT-06-006, ITEM-1 and ITEM-2. The concentration of each phagepreparation was adjusted to give an approximately equal reactivity withMAb 9E10, to normalize the amount of displayed hFn14 in each phagepreparation. Each normalized preparation was 10-fold serially dilutedand added in solution to the MAbs attached to the solid phase. AllELISAs shown were performed simultaneously with the same preparations ofphage. Binding was detected using HRP-conjugated anti-M13 phagepolyclonal IgG and TMB substrate. Optical density at 450 nm wasquantified using a Spectramax spectrophotometer. Error bars for eachdilution represent the standard deviation of duplicate assays. In thisfigure, reference to “FN14 Ecto” is a reference to the extracellulardomain of hFn14. Methods according to Epitope mapping using phageexpressed Fn14—Method 1.

FIG. 18B is a series of graphical representations showing results ofELISAs using the same phage constructs as in FIG. 18A and showing thatnone of the phage-displayed hFn14 constructs bind significantly to thenegative control MAb 5G8. ELISAs were performed under similar conditionsto those described for FIG. 16A. In this figure, reference to “FN14Ecto” is a reference to the extracellular domain of hFn14.

FIG. 19 is a series of graphical representations showing anti-Fn14monoclonal antibody CRCBT-06-007 binds specifically to sub-domain 2 ofthe hFn14 extracellular domain. Panel A Results of ELISAs are depictedshowing the reactivity of the phage-displayed hFn14 extracellular domain(Fn14 Ecto), sub-domain 1 (SD1) and sub-domain 2 (SD2) with theanti-Fn14 monoclonal antibody CRCBT-06-007. The concentration of eachphage preparation was adjusted to give an approximately equal reactivitywith MAb 9E10, to normalize the amount of displayed hFn14 in each phagepreparation. Each normalized preparation was 10-fold serially dilutedand added in solution to the MAbs attached to the solid phase. ELISAsshown here were performed simultaneously with the same preparations ofphage. Binding was detected using HRP-conjugated anti-M13 phagepolyclonal IgG and TMB substrate. Optical density at 450 nm wasquantified using a Spectramax spectrophotometer. Error bars for eachdilution represent the standard deviation of duplicate assays. Panel Bshows results of ELISAs using the same phage constructs as in FigurePanel A and showing that none of the phage-displayed hFn14 constructsbind significantly to the negative control MAb 5G8. ELISAs wereperformed under similar conditions to those described for Panel A.Methods according to Epitope mapping using phage expressed Fn14—Method1.

FIG. 20 is a series of schematic representations. Panel A. shows theFn14 extracellular domain, Panel B. shows the Fn14 sub-domain 1p (alonger form of sub-domain 1 expressed on phage), Panel C. shows the Fn14sub-domain 2 and Panel D. shows the Fn14 sub-domain 3. Panel E. showsthe Fn14 sub-domain 2 in which the third and sixth cysteine residues inFn14 ECD that form disulfide bonds are mutated to serine (designatedherein “Sub-domain 2 Cys 3&6 ΔS”). Panel F. shows the Fn14 sub-domain 2in which the fourth and fifth cysteine residues in Fn14 ECD that formdisulfide bonds are mutated to serine (designated herein “Sub-domain 2Cys 4&5 ΔS”). All panels show disulfide connectivity according to thesolution structure (He et al., 2009). Bold large case “S” representsserine residue substituted in place of naturally-occurring cysteineresidue.

FIG. 21A is a graphical representation showing specificity of anti-Fn14mAbs. Anti-Fn14 mAbs CRCBT-06-001, CRCBT-06-002, CRCBT-06-003,CRCBT-06-004, CRCBT-06-005, CRCBT-06-006 and CRCBT-06-007 are reactivewith sub-domains 2 and 3 but not sub-domain 1. Very low reactivity tothe control peptide was observed for all anti-Fn14 mAbs. The controlpeptide was also reactive with its cognate (isotype control) antibody.

FIG. 21B is a graphical representation showing reactivity of anti-Fn14mAbs CRCBT-06-001, CRCBT-06-002, CRCBT-06-003, CRCBT-06-004,CRCBT-06-005, CRCBT-06-006, CRCBT-06-007 and ITEM-1 with a syntheticpeptide representing sub-domain 2 of Fn14. Plates were coated withsub-domain 2 peptide and various dilutions of antibody were allowed tobind, bound antibody was detected with anti-mouse HRP and TMB substrate.The average of duplicate readings are plotted, error bars representstandard deviations. The ELISA was repeated to ensure consistentresults. The vertical line at 5 ng/ml within the linear part of thecurves is the point at which a comparison of the reactivity of each mAbwas analyzed.

FIG. 21C is a graphical representation showing reactivity of anti-Fn14mAbs CRCBT-06-001, CRCBT-06-002, CRCBT-06-003, CRCBT-06-004,CRCBT-06-005, CRCBT-06-006, CRCBT-06-007 and ITEM-1 with a syntheticpeptide representing sub-domain 3 of Fn14. Plates were coated withsub-domain 3 peptide and various dilutions of antibody were allowed tobind, bound antibody was detected with anti-mouse HRP and TMB substrate.The average of duplicate readings are plotted, error bars representstandard deviations. The ELISA was repeated to ensure consistentresults.

FIG. 22 is a graphical representation showing the reactivity of hFn14phage constructs with mAb 9E10 control (Panel i), CRCBT-06-002 (PanelII) and ITEM-1 (Panel iii) to phage displayed fragments of Fn14 andmutants thereof. Binding was detected using HRP-conjugatedanti-M13-phage antibody and TMB substrate. Optical density at 450 nm wasquantified using a Spectramax plate reader. Error bars for each dilutionrepresent the ranges of duplicate values. Methods according to Epitopemapping using phage expressed Fn14—Method 1

FIG. 23A is a graphical representation showing the reactivity of hFn14with the anti-Fn14 monoclonal antibodies (as indicated in the Figure) asdetermined using ELISA. Binding was detected using HRP-conjugatedanti-mouse IgG and TMB substrate. Optical density at 450 nm wasquantified using a Spectramax spectrophotometer. Antibody concentrationin μg/ml.

FIGS. 23B and C are graphical representations showing results of similarELISAs as in FIG. 23A, however using mouse Fn14 to anti-Fn14 monoclonalantibodies (as indicated). Antibody concentration in μg/ml. includes aseries of graphical representations showing binding of antibodies CRCBT-

FIGS. 24A-F are graphical representations showing binding of hFn14mutants mutants R56P, R56A and R56K to anti-Fn14 antibodies. FIGS. 24Aand 24B show results of ELISAs showing the reactivity of hFn14 mutantR56P with the nti-Fn14 monoclonal antibodies (as indicated in theFigure). Binding was detected using HRP-conjugated anti-mouse IgG andTMB substrate. Optical density at 450 nm was quantified using aSpectramax spectrophotometer. FIGS. 24C and D show results of ELISAsperformed comparing the binding of hFn14 mutant R56A to anti-Fn14monoclonal antibodies (as indicated). FIGS. 24E and F show results ofELISAs performed comparing the binding of hFn14 mutant R56K to anti-Fn14monoclonal antibodies (as indicated). Antibody concentration in μg/ml.

FIGS. 25A and B are graphical representations showing binding ofpurified hFn14 mutants R58A to anti-Fn14 antibodies (as indicated in theFigure). Binding was detected using HRP-conjugated anti-mouse IgG andTMB substrate. Optical density at 450 nm was quantified using aSpectramax spectrophotometer. Antibody concentration in μg/ml.

FIG. 26 is a graphical representation showing results of an ELISAtesting the reactivity of mutant hFn14 phage construct comprising a R58Amutation with the anti-Fn14 monoclonal antibodies CRCBT-06-001,CRCBT-06-002, CRCBT-06-004 compared to ITEM-1 and ITEM-4. Binding wasdetected using Biotin-conjugated anti-M13-phage monoclonal antibodyfollowed by HRP-conjugated Streptavidin and TMB substrate. Opticaldensity at 450 nm was quantified using a Spectramax spectrophotometer.Error bars for each dilution represent the standard deviation ofduplicate assays. X axis=dilution of phage. Methods according to Epitopemapping using phage expressed Fn14—Method 2

FIGS. 27A to H are a series of graphical representations showing bindingof phage-displayed mutant hFn14 constructs to anti-Fn14 mAbs. Resultsare depicted of ELISAs testing the reactivity of mutant hFn14 phageconstructs with the anti-Fn14 monoclonal antibodies CRCBT-06-001,CRCBT-06-002, CRCBT-06-004 compared to ITEM-1 and ITEM-4 and in somecases ITEM-2. For mutants P59A and F63A, binding was detected usingHRP-conjugated anti-M13-phage polyclonal IgG and TMB substrate. For allother mutants binding was detected using Biotin-conjugatedanti-M13-phage monoclonal antibody followed by HRP-conjugatedStreptavidin and TMB substrate. Optical density at 450 nm was quantifiedusing a Spectramax spectrophotometer. Error bars for each dilutionrepresent the standard deviation of duplicate assays. FIG. 27A. wildtype Fn14, FIG. 27B. W42A, FIG. 27C. S54A, FIG. 27D. A57G, FIG. 27E.P59A, FIG. 27F. S61A, FIG. 27G. F63A, FIG. 27H. L65A. In all cases errorbars represent the standard deviation. X axis=dilution of phage. Methodsaccording to Epitope mapping using phage expressed Fn14—Method 2

FIGS. 28A to C are a series of graphical representations showing bindingof phage-displayed mutant hFn14 constructs to anti-Fn14 mAbs. ELISAsshowing the reactivity of mutant hFn14 phage constructs with theanti-Fn14 monoclonal antibodies CRCBT-06-001, CRCBT-06-002, CRCBT-06-004compared to ITEM-1 and ITEM-4. Binding was detected usingBiotin-conjugated anti-M13-phage monoclonal antibody followed byHRP-conjugated Streptavidin and TMB substrate. Optical density at 450 nmwas quantified using a Spectramax spectrophotometer. Error bars for eachdilution represent the standard deviation of duplicate assays. FIG. 28A.L46A, FIG. 28B. D51A and FIG. 28C. D62A. In all cases error barsrepresent the standard deviation. X axis=dilution of phage. Methodsaccording to Epitope mapping using phage expressed Fn14 —Method 2.

FIGS. 29A and 29B are graphical representations showing binding ofphage-displayed mutant hFn14 constructs to anti-Fn14 mAbs. ELISAsshowing the reactivity of mutant H60A and H60K hFn14 phage constructswith the anti-Fn14 monoclonal antibodies CRCBT-06-001, CRCBT-06-002,CRCBT-06-004 compared to ITEM-1 and ITEM-4. ITEM-2 was assessed on H60K.Binding was detected using Biotin-conjugated anti-M13-phage monoclonalantibody followed by HRP-conjugated Streptavidin and TMB substrate.Optical density at 450 nm was quantified using a Spectramaxspectrophotometer. Error bars for each dilution represent the standarddeviation of duplicate assays. X axis=dilution of phage. Methodsaccording to Epitope mapping using phage expressed Fn14—Method 2.

FIGS. 30A and 30B are graphical representations showing binding ofanti-Fn14 antibodies to disulfide bond mutant peptides. FIG. 30A showsresults of ELISAs in which anti-Fn14 antibodies were titrated(0.1-0.0001 μg/ml) and binding to sub-domain 2 cys3&6ΔS mutant wasassessed. FIG. 30B is similar to FIG. 27A, however binding to sub-domain2 cys4&5ΔS mutant was assessed (1-0.0001 μg/ml).

FIG. 31 is a series of graphical representations showing staining of invitro cultured cells for Fn14 expression levels. Cells were cultured inthe presence or absence of 100 nM 4-OHT for 24 hours (Panel A showsresults for MEF SV40v12Hras Fn14, Panel B shows results for MEF SV40 andPanel C shows results for Lewis lung carcinoma). Cells were harvestedand stained for hFn14 expression using a commercially availableanti-Fn14 antibody followed by an anti-mouse Alexafluor-647 antibody fordetection by flow cytometry. Staining with anti-Fn14 indicated by solidtrace, secondary antibody control staining by hashed trace and unstainedcells by dotted trace.

FIG. 32 is a series of graphical representations showing analysis ofcytokine secretion by MEF tumor cell lines. MEF tumor cell linescontaining Fn14 or Fn14-GPI were induced with 4-OHT or uninduced. After48 hours under normal growth condition the media from cells washarvested and the levels of IL-6 were assessed using BD CBA MouseInflammation Kit and quantitative analysis performed using FCAP Arraysoftware (BD Biosciences).

FIG. 33 is a series of representations showing creation of an Fn14 invivo tumor model. Female C57BL/6 mice were injected with tumor cells onday 1. Panel A is a graphical representation showing results ofmeasurements taken as tumors formed and the tumor volume for each mousewas calculated ([length*width²]/2) and group averages were graphed.Panel B is a graphical representation showing results of analysis inwhich the body weight for each mouse assessed daily and standardizedagainst day 1 weight as 100. The average for each group was calculatedand graphed. Groups include tumors containing wildtype hFn14 (v12HrasFn14; ♦; n=6) or parent tumor cell line (v12Hras; ▪; n=6). Experimentswere repeated reproducibly a number of times, data from one experimentpresented. Error bars represent the SEM. Note—Panel B Y axis does notbegin at 0.

FIG. 34A is a series of schematic representation of the sequence of theextracellular region of human Fn14 (BOLD) fused to the GPI anchor codingregion of TrailR3 (ITALICS) to create hFn14-GPI. The predicted GPIattachment serine residue is indicated in bold text with an arrow.Underlined region indicates the Fn14 signal sequence.

FIG. 34B includes two graphical representations showing results ofintroducing hFn14-expressing tumors or non-signaling hFn14-expressingtumors into mice. Female C57BL/6 mice were injected with tumor cells onday 1. Groups include tumors containing wildtype Fn14 (♦; n=14) orFn14-GPI tumors (▪; n=14). Panel i shows results of analysis performedas tumors formed, and measurements were taken and the tumor volume foreach mouse was calculated ([length*width²]/2). Panel ii shows results ofanalysis in which the body weight for each mouse. The group averageswere calculated and graphed over time. Note—Panel ii Y axis does notbegin at 0. Data was graphed as group averages. Experiments wererepeated reproducibly a number of times, data from one representativeexperiment are presented. Error bars represent the SEM.

FIG. 35 includes two graphical representations showing effects of tumorsconstitutively expressing human Fn14. Female C57BL/6 mice were injectedwith tumor cells on day 1. Groups include tumors containing low levelFn14 (n=3, mice 137-139) and constitutively expressed Fn14 (n=6, mice149-154) as indicated beside legend. Panel A shows results of analysisin which the body weight for each mouse was assessed daily and graphedover time. Note—Panel A Y axis does not begin at 0. Panel B showsresults of measurements of tumor volume. Measurements were taken and thetumor volume for each mouse was calculated ([length*width²]/2) andgraphed individually over time.

FIG. 36 is a graphical representation showing results of treatment ofhFn14 tumors with CRCBT-06-001. Female C57BL/6 mice were injected witheither hFn14 or hFn14-GPI tumor cells on day 1. On day 6 each group wassegregated evenly and half were treated twice a week with an IPinjection of CRCBT-06-001 (10 mg/kg) for a total of 4 weeks. The otherhalf of the group received no treatment. Body weight was measured daily,standardized against starting weight as 100 and group averages weregraphed. ▪=Fn14 antibody treated, ♦=Fn14 untreated, ●=Fn14-GPI antibodytreated, ▴=Fn14-GPI untreated. Treatment days are indicated by a blackdownward arrow (↓). Upward pointing arrows indicate a mouse waseuthanized, the symbol underneath indicating from which group. Eachgroup n=3 and the error bars represent SEM. Note—Y axis does not beginat 0.

FIG. 37A is a graphical representation showing results of treatment ofhFn14 tumor mice with CRCBT-06-001, CRCBT-06-002, CRCBT-06-004 orITEM-1. Female C57BL/6 mice were injected with Fn14 tumor cells onday 1. On day 7, groups of mice (n=6) were given a single IP injectionof purified antibody (5 mg/kg) or no treatment. A. Body weight wasmeasured daily and graphed as weight standardised against startingweight as 100% and graphed as group average. Treatment day indicated bya black downward arrow (↓). Error bars represent SEM. Note—Y axis doesnot cross at 0.

FIG. 37B includes two graphical representations showing survival ofhFn14 tumor mice with CRCBT-06-001, CRCBT-06-002, CRCBT-06-004 orITEM-1. The left-hand panel indicates the day each mouse was killed andthe group average survival day. Note * indicates a data point representshealthy mice with no weight loss at the termination of the experiment(day 27). Hashed line (- -) represents antibody treatment (day 7). Theright-hand panel is a Kaplan-Meier curve showing survival.

FIG. 38 is a graphical representation showing CRCBT-06-001 dose responseand rescue of mice from weight loss and illness. Female C57BL/6 micewere injected with hFn14 tumor cells on day 1. On day 6 groups weretreated with a single IP injection of CRCBT-06-001 (0-10 mg/kg).Untreated mice received a vehicle injection (PBS). On day 9 or 10 asindicated, mice exhibiting obvious weight loss were treated with asingle IP injection of 10 mg/kg CRCBT-06-001. Body weight was measureddaily, standardized against the starting weight (day 1) as 100 and groupaverages were graphed. The standardized weights for each group weregraphed as group averages. Error bars represent the SEM. Antibody dosetreatment day is indicated by a black downward arrow. Each group n=3.Symbols represent a mouse was euthanized from the group designated withthat symbol. Note—Y axis does not begin at 0.

FIG. 39A is a graphical representation showing effects of CRCBT-06-001on body mass in mice injected with MEF Fn14 cells. On day 1, female 11week old C57BL/6 mice were given a single subcutaneous injection of MEFFn14 or with the parent cell line (Hras). On day 6, mice were given asingle intraperitoneal injection of IgG2b isotype control antibody(Hras+IgG2b, MEF Fn14+IgG2b, n=8/group) or CRCBT-06-001 (MEF Fn14+001,n=8). Body mass was measured on days 1 and 11. Data are means±SEM.*P<0.01 vs. Hras+IgG2b; †P<0.05 vs. MEFFn14+IgG2b.

FIG. 39B is a graphical representation showing effects of CRCBT-06-001on tumor-free body mass in mice injected with MEF Fn14 cells. On day 11,the tumor from mice described in relation to FIG. 28A was surgicallyexcised and weighed allowing calculation of the percentage change intumor-free body mass from pre-inoculation (B). Data are means±SEM.*P<0.01 vs. Hras+IgG2b; †P<0.05 vs. MEFFn14+IgG2b.

FIG. 40A is a graphical representation showing effects of CRCBT-06-001on tumor mass in mice injected with MEF Fn14 cells. On day 11, the tumorfrom mice described in relation to FIG. 39A was surgically excised andweighed allowing calculation of the percentage change in tumor mass.

FIG. 40B is a graphical representation showing effects of CRCBT-06-001on tumor volume in mice injected with MEF Fn14 cells. On day 11, thetumor from mice described in relation to FIG. 39A was surgically excisedand tumor volume calculated.

FIGS. 41A and B are a series of graphical representations showingeffects of CRCBT-06-001 on muscle mass in mice injected with MEF Fn14cells. On day 11, selected hindlimb muscles (as indicated in FIG. 41A);and epididymal fat and the heart (as indicated in FIG. 42B) were excisedand weighed on an analytical balance. EDL, extensor digitorum longus;Plant, plantaris; TA, tibialis anterior; Gastroc, gastrocnemius; Quad,quadriceps. Data are means±SEM. *P<0.05 vs. Hras+IgG2b; †P<0.05 vs. MEFFn14+IgG2b.

FIGS. 42A and B are graphical representations showing effects ofCRCBT-06-001 on whole body strength in mice injected with MEF Fn14cells. On day 11, whole body strength was assessed using a grip strengthmeter and expressed in absolute values (FIG. 42A) and normalised to bodymass (FIG. 42B). Data are means±SEM.

FIGS. 43A-D are graphical representations showing effects ofCRCBT-06-001 effects on functional properties of tibialis anterior (TA)muscles in situ in mice injected with MEFFn14. On day 11, peak twitchforce (FIG. 43A), peak tetanic force (FIG. 43B), frequency-forcerelationship (FIG. 43C) and force production during and following 4minutes of fatiguing intermittent stimulation (FIG. 43D) was assessed inTA muscles in situ. Data are means±SEM. ^(a)P<0.05 main effectHras+IgG2b vs MEF Fn14+IgG2b.

FIG. 44 is a series of graphical representations showing effects ofCRCBT-06-001 on fibre size, fibre type composition and fibre oxidativeenzyme capacity in tibialis anterior (TA) muscles from mice injectedwith MEF Fn14 cells. On day 11, TA muscles were excised and frozen forsubsequent histological analyses. Quantification of laminin, N2.261 andSDH based on reaction intensity facilitated determination of theproportion of type IIa and type IIx/b fibres (non-N2.261 reactingfibres) (left panel), the area of the type IIa and type IIx/b fibres(center panel) and the SDH activity based on reaction intensity of thetype IIa and type IIx/b fibres (right panel) are shown. Data aremeans±SEM. *P<0.05 vs. Hras+IgG2b; †P<0.05 vs. MEF Fn14+IgG2b.

FIGS. 45A and B are graphical representations showing the effect ofCRCBT-06-002 effect on Colon-26 model for cancer cachexia. Male 11 weekold CD2F1 mice were inoculated with 1×10⁶ cells s/c in the flank on day1 to form solid tumors. Groups included Colon-26 Untreated (n=5),Colon-26+CRCBT-06-002 treated (n=5), Pair fed (n=4) and non-tumoruntreated controls (n=3). On days 5, 12, 15 and 20, mice were treatedwith an IP injection of 10 mg/kg CRCBT-06-002 (i). Untreated micereceived no treatment. For FIG. 45A, body mass was measured daily andgraphed as group average weights standardised against starting weight(day 1) as 100%. For FIG. 45B daily survival after initiation ofantibody treatment (day 5) was graphed as a Kaplan-Meier curve. Note—twoantibody-treated mice were killed due to maximal tumor volume endpointand therefore not included in this graph.

FIGS. 46A and B are graphical representations showing the effect ofCRCBT-06-002 effect on tumors in the Colon-26 model for cancer cachexia.For FIG. 46A, as tumors formed, measurements were taken and the tumorvolume for each mouse was calculated ([length*width²]/2). Data wasgraphed group averages Error bars represent the SEM. The apparent dropin tumour volume (from about 1200 to 400 mm³) at day 20 reflects thereduction in group number due to killing of a number of mice that hadreached the maximum allowed tumour size. The surviving mice at 21 hadaverage tumour volume of about 400 mm³. For FIG. 46B, female 9-10 weekold Balb/c mice were inoculated with 1×10⁶ cells s/c in the flank on day1 to form solid tumors. Groups included Untreated (n=5) and CRCBT-06-002treated (n=5). On days 8, 12, 15, 19 and 22, mice were treated with anIP injection of 10 mg/kg CRCBT-06-002 as indicated. As tumors formed,measurements were taken and the tumor volume for each mouse wascalculated. Data is graphed for group averages. Error bars represent theSEM.

FIGS. 47A to C are graphical representations showing the effect ofCRCBT-06-001 on CD2F1 mice administered Colon-26 cells. Male 12 week oldCD2F1 mice were inoculated with 1×10⁶ cells s/c in the flank on day 1 toform solid tumors. Groups included Untreated (n=5; mice numbers 1-5) andCRCBT-06-001 treated (n=5; mice numbers 6-10). On days 16 and 20, micewere treated with an IP injection of 10 mg/kg CRCBT-06-001. Antibodytreatment days as indicated ↓. Data was graphed standardized againststarting body mass as 100% for group averages (shown in FIG. 47A) Errorbars represent the SEM. FIG. 47B shows survival graphed as aKaplan-Meier curve. FIG. 47C shows group averages of tumor volume. Errorbars represent the SEM.

FIGS. 48 A to C are graphical representations showing the effects ofCRCBT-06-002 on body mass in the Colon-26 model of cancer cachexia. Male11 week old CD2F1 mice were inoculated with 1×10⁶ cells s/c in the flankon day 1 to form solid tumors. Groups included Untreated (n=9), IgGtreated pair-fed (n=10) and CRCBT-06-002 treated pair-fed (n=10). Ondays 8, 12 and 16, mice were treated with an IP injection of 10 mg/kgIgG or 10 mg/kg CRCBT-06-002 (i). Body mass was measured daily andexpressed as absolute body mass (and is shown in FIG. 48A) and body massnormalised to starting weight as 100. Note: Y axis does not begin at 0(FIG. 48B). On day 22, the tumor was surgically excised and weighedallowing calculation of the percentage change in tumor-free body massfrom pre-inoculation (FIG. 48C). Data are means±SEM. *P<0.05 vs. C-26;↓P<0.05 vs. C-26+IgG.

FIGS. 49A to C are graphical representations showing the effect ofCRCBT-06-002 on tumor size in the Colon-26 model of cancer cachexia.Tumor size was measured daily with digital calipers (results depicted inFIG. 49A). On day 22, the tumor was surgically excised and weighed(results depicted in FIG. 49B), and normalised to total body mass (asshown in FIG. 49C). Data are means±SEM. *P<0.05 vs. C-26; †P<0.05 vs.C-26+IgG

FIGS. 50A and B are graphical representations showing the effects ofCRCBT-06-002 on muscle mass in the Colon-26 model of cancer cachexia. Onday 22, selected hindlimb muscles (FIG. 50A), epididymal fat and theheart (FIG. 50B) were excised and weighed on an analytical balance. EDL,extensor digitorum longus; Plant, plantaris; TA, tibialis anterior;Gastroc, gastrocnemius; Quad, quadriceps. Data are means±SEM. *P<0.05vs. C-26; †P<0.05 vs. C-26+IgG.

FIGS. 51A and B are graphical representations showing the effects ofCRCBT-06-002 on whole body strength and mobility in the Colon-26 modelof cancer cachexia. On day 21, whole body strength was assessed using agrip strength meter (FIG. 51A) and whole body mobility was assessed vialatency-to-fall during a rotarod test (FIG. 51B). Data are means±SEM.*P<0.05 vs. C-26; †P<0.05 vs. C-26+IgG.

FIGS. 52A to D are graphical representations showing the effect ofCRCBT-06-002 effects on functional properties of tibialis anterior (TA)muscles in situ in the Colon-26 model of cancer cachexia. On day 22,peak twitch force (FIG. 52A), peak tetanic force (FIG. 52B),frequency-force relationship (FIG. 52C) and force production during andfollowing 4 minutes of fatiguing intermittent stimulation (FIG. 52D) wasassessed in TA muscles in situ. Data are means±SEM. *P<0.01 vs. C-26;†P<0.05 vs. C-26+IgG; T<0.03 main effect C-26+002 higher than C-26+IgG.

FIGS. 53A to C are graphical representations showing the effects ofCRCBT-06-002 on muscle fibre architecture, fibre size, fibre typecomposition and fibre oxidative enzyme capacity in tibialis anterior(TA) muscles from the Colon-26 model of cancer cachexia. On day 22, TAmuscles were excised and frozen for subsequent analyses. Quantificationof laminin and N2.261 reactions enabled assessment of thecross-sectional area (CSA) of type IIa and type IIx/b fibres as well asaverage muscle fibre CSA (FIG. 53A). It also enabled assessment of theproportion of type IIa and type IIx/b muscle fibres (FIG. 53B).Quantification of SDH and N2.261 allowed assessment of the oxidativeenzyme capacity of type IIa and type IIx/b fibres as well as averagemuscle fibre oxidative enzyme capacity (FIG. 53C). *P<0.05 vs. C-26;†P<0.05 vs. C-26+IgG.

FIG. 54 is a graphical representation showing that administration of ananti-Fn14 antibody post diabetes onset partially prevents weight lossassociated with streptozotocin (STZ)-induced diabetes. Diabetes wasinduced in male C57Bl/6 mice by multiple low dose STZ administration.Groups included STZ, Vehicle, Pair-Fed and 20 mg/kg CRCBT-06-002.↓=Antibody treatment day. Daily body weight measurements werestandardised against the starting weight (day 1) as 100% and the groupaverages were graphed. n=8, Error bars represent SE. Note Y axis doesnot cross at 0.

FIG. 55 is a graphical representation showing Cumulative water intake inmice depicted in FIG. 54. Water intake was measured daily for each group(n=8) and graphed as cumulative water intake.

FIG. 56 is a graphical representation showing blood glucose analysis ofmice treated as described in respect of FIG. 54. Blood glucose wasassessed in non-fasted mice on the final day of the experiment using anAccu-check blood glucose monitor and graphed as group average. Errorbars represent SEM. When a reading out of range was detected, themaximum possible reading of 600 mg/dl was assigned. n=8 per group

FIG. 57 is a series of graphical representations showing anti-Fn14antibody administration post diabetes onset attenuates muscle wastingassociated with STZ induced diabetes. Diabetes was induced in maleC57Bl/6 mice by multiple low dose STZ administration as in FIG. 54.Groups included STZ, Vehicle, Pair-Fed and 20 mg/kg CRCBT-06-002antibody treatment. Antibody treatment was administered on day 7. PanelA shows group average tissue mass was calculated and graphed. Tissuemass was standardized to final (Panel B) and starting body mass (PanelC) and graphed as group averages, Panels B and C respectively show massof i. Quadricep ii. Heart iii. Tibialis anterior iv. Epididymal fat.Error bars represent the SEM. n=8. Note some Y axis do not cross at 0.

KEY TO SEQUENCE LISTING

-   SEQ ID NO 1: amino acid sequence of human Fn14-   SEQ ID NO 2: amino acid sequence of Fn14 extracellular domain-   SEQ ID NO 3: amino acid sequence of Fn14 gene fragment RW129-   SEQ ID NO 4: amino acid sequence of Fn14 gene fragment RW131-   SEQ ID NO 5: amino acid sequence of Fn14 gene fragment RW127-   SEQ ID NO 6: amino acid sequence of Fn14 gene fragment RW125-   SEQ ID NO 7: amino acid sequence of Fn14 gene fragment RW120-   SEQ ID NO 8: amino acid sequence of Fn14 gene fragment RW118-   SEQ ID NO 9: amino acid sequence of Fn14 gene fragment RW114    SEQ ID NO 10: amino acid sequence of Fn14 gene fragment RW98-   SEQ ID NO 11: amino acid sequence of Fn14 gene fragment RW95-   SEQ ID NO 12: amino acid sequence of Fn14 gene fragment RW91-   SEQ ID NO 13: amino acid sequence for consensus sequence for V_(H)-   SEQ ID NO 14: amino acid sequence for consensus sequence for V_(L)-   SEQ ID NO 15: amino acid sequence of V_(H) of CRCBT-06-001-   SEQ ID NO 16: amino acid sequence of V_(H) of CRCBT-06-002-   SEQ ID NO 17: amino acid sequence of V_(H) of CRCBT-06-003-   SEQ ID NO 18: amino acid sequence of V_(H) of CRCBT-06-004-   SEQ ID NO 19: amino acid sequence of V_(H) of CRCBT-06-005-   SEQ ID NO 20: amino acid sequence of V_(H) of CRCBT-06-006-   SEQ ID NO 21: amino acid sequence of V_(H) of CRCBT-06-007-   SEQ ID NO 22: amino acid sequence of V_(L) of CRCBT-06-001-   SEQ ID NO 23: amino acid sequence of V_(L) of CRCBT-06-002-   SEQ ID NO 24: amino acid sequence of V_(I), of CRCBT-06-003    SEQ ID NO 25: amino acid sequence of V_(L) of CRCBT-06-004-   SEQ ID NO 26: amino acid sequence of V_(L) of CRCBT-06-005-   SEQ ID NO 27: amino acid sequence of V_(L) of CRCBT-06-006-   SEQ ID NO 28: amino acid sequence of V_(L) of CRCBT-06-007-   SEQ ID NO 29: amino acid sequence of hFn14 extracellular domain    mutant construct D45A-   SEQ ID NO 30: amino acid sequence of hFn14 extracellular domain    mutant construct K48A-   SEQ ID NO 31: amino acid sequence of hFn14 extracellular domain    mutant construct M50A-   SEQ ID NO 32: amino acid sequence of hFn14 extracellular domain    mutant construct D62E-   SEQ ID NO: 33: amino acid sequence of hFn14 subdomain 1-   SEQ ID NO 34: amino acid sequence of hFn14 subdomain 2-   SEQ ID NO: 35: amino acid sequence of hFn14 subdomain 3-   SEQ ID NO 36: amino acid sequence of Fn14-GPI control-   SEQ ID NO 37: nucleotide sequence of a primer for amplifying nucleic    acid encoding extracellular domain of Fn14.-   SEQ ID NO 38: nucleotide sequence of a primer for amplifying nucleic    acid encoding extracellular domain of Fn14.-   SEQ ID NO 39: nucleotide sequence of a primer for amplifying Trail    R3 GPI anchor coding region.-   SEQ ID NO 40: nucleotide sequence of a primer for amplifying Trail    R3 GPI anchor coding region.-   SEQ ID NO 41: nucleotide sequence of a primer for amplifying light    chain variable region of an antibody.-   SEQ ID NO 42: nucleotide sequence of a primer for amplifying light    chain variable region of an antibody.-   SEQ ID NO 43: nucleotide sequence of a primer for amplifying heavy    chain variable region of an antibody.-   SEQ ID NO 44: nucleotide sequence of a primer for amplifying heavy    chain variable region of an antibody.-   SEQ ID NO 45: nucleotide sequence of a primer for amplifying heavy    chain variable region of an antibody.-   SEQ ID NO: 46: amino acid sequence of sub-domain 1p.-   SEQ ID NO: 47 Fn14 sub-domain 2 in which the third and sixth    cysteine residues in Fn14 ECD that form disulfide bonds are mutated    to serine (designated herein “Sub-domain 2 Cys 3&6 ΔS”).    SEQ ID NO: 48: Fn14 sub-domain 2 in which the fourth and fifth    cysteine residues in Fn14 ECD that form disulfide bonds are mutated    to serine (designated herein “Sub-domain 2 Cys 4&5 ΔS”).-   SEQ ID NO: 49: Fn14 T33N mutant-   SEQ ID NO: 50: Fn14 A34S mutant-   SEQ ID NO: 51: Fn14 R38S mutant-   SEQ ID NO: 52: Fn14 R56P mutant-   SEQ ID NO: 53: Fn14 L77M mutant-   SEQ ID NO: 54: Fn14 R56A mutant-   SEQ ID NO: 55: Fn14 R56K mutant-   SEQ ID NO: 56: Fn14 R58A mutant-   SEQ ID NO: 57: Fn14 W42A mutant-   SEQ ID NO: 58: Fn14 L46A mutant-   SEQ ID NO: 59: Fn14 D51A mutant-   SEQ ID NO: 60: Fn14 S54A mutant-   SEQ ID NO: 61: Fn14 A57G mutant-   SEQ ID NO: 62: Fn14 P59A mutant-   SEQ ID NO: 63: Fn14 H60A mutant-   SEQ ID NO: 64: Fn14 S61A mutant-   SEQ ID NO: 65: Fn14 D62A mutant-   SEQ ID NO: 66: Fn14 F63A mutant-   SEQ ID NO: 67: Fn14 L65A mutant-   SEQ ID NO: 68: Fn14 H60K mutant-   SEQ ID NO: 69: Primer for amplifying antibody light chain-   SEQ ID NO: 70: Primer for amplifying antibody light chain

DETAILED DESCRIPTION

General

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

Those skilled in the art will appreciate that the present disclosure issusceptible to variations and modifications other than thosespecifically described. It is to be understood that the disclosureincludes all such variations and modifications. The disclosure alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specificexamples described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the disclosure.

Any example of the present disclosure herein shall be taken to applymutatis mutandis to any other example of the disclosure unlessspecifically stated otherwise.

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (for example, in cellculture, molecular genetics, immunology, immunohistochemistry, proteinchemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present disclosure are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as,Perbal (1984), Sambrook et al., (1989), Brown (1991), Glover and Hames(1995 and 1996), and Ausubel et al., (1988, including all updates untilpresent), Harlow and Lane, (1988), Coligan et al., (including allupdates until present) and Zola (1987).

The description and definitions of variable regions and parts thereof,immunoglobulins, antibodies and fragments thereof herein may be furtherclarified by the discussion in Kabat, 1987 and/or 1991, Bork et al.,1994 and/or Chothia and Lesk, 1987 and/or 1989 or Al-Lazikani et al.,1997.

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

As used herein the term “derived from” shall be taken to indicate that aspecified integer may be obtained from a particular source albeit notnecessarily directly from that source.

In one example of the present disclosure, the X at position 103 is aglycine. In one example of the present disclosure, the X at position 103is a glutamate or glutamic acid.

SELECTED DEFINITIONS

As used herein, the term “Fn14” collectively refers to Fn14 from allmammals, such as from humans and from rodents. The term “hFn14” or“human Fn14” refers to Fn14 from humans. For the purpose of nomenclatureand not limitation, an amino acid sequence of an hFn14 is set forth inSEQ ID NO: 1.

For the purposes for the present disclosure, the term “antibody”includes a protein capable of specifically binding to one or a fewclosely related antigens (e.g., Fn14) by virtue of a Fv. This termincludes four chain antibodies (e.g., two light chains and two heavychains), recombinant or modified antibodies (e.g., chimeric antibodies,humanized antibodies, primatized antibodies, de-immunized antibodies andhalf antibodies, bispecific antibodies). An antibody generally comprisesconstant domains, which can be arranged into a constant region orconstant fragment or fragment crystallizable (Fc). Exemplary forms ofantibodies comprise a four-chain structure as their basic unit.Full-length antibodies comprise two heavy chains (˜50-70 kDa) covalentlylinked and two light chains (˜23 kDa each). A light chain generallycomprises a variable region and a constant domain and in mammals iseither a κ light chain or a λ light chain. A heavy chain generallycomprises a variable region and one or two constant domain(s) linked bya hinge region to additional constant domain(s). Heavy chains of mammalsare of one of the following types α, δ, ε, γ, or μ. Each light chain isalso covalently linked to one of the heavy chains. For example, the twoheavy chains and the heavy and light chains are held together byinter-chain disulfide bonds and by non-covalent interactions. The numberof inter-chain disulfide bonds can vary among different types ofantibodies. Each chain has an N-terminal variable region (V_(H) or V_(L)wherein each are ˜110 amino acids in length) and one or more constantdomains at the C-terminus. The constant domain of the light chain (C_(L)which is ˜110 amino acids in length) is aligned with and disulfidebonded to the first constant domain of the heavy chain (C_(H) which is−330-440 amino acids in length). The light chain variable region isaligned with the variable region of the heavy chain. The antibody heavychain can comprise 2 or more additional C_(H) domains (such as, C_(H)2,C_(H)3 and the like) and can comprise a hinge region can be identifiedbetween the C_(H)1 and C_(H)2 constant domains. Antibodies can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁ and IgA₂) or subclass. In one example, the antibody isa murine (mouse or rat) antibody or a humanized form thereof or aprimate (such as, human) antibody.

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody in its substantiallyintact form, as opposed to an antigen binding fragment of an antibody.Specifically, whole antibodies include those with heavy and light chainsincluding a constant region. The constant region may be wild-typesequence constant regions (e.g., human wild-type sequence constantregions) or amino acid sequence variants thereof.

The term “Fn14-binding protein” shall be taken to include a singlepolypeptide chain, (i.e., a series of contiguous amino acids linked bypeptide bonds), or a series of polypeptide chains covalently ornon-covalently linked to one another (i.e., a polypeptide complex)capable of binding to Fn14 in the manner described and/or claimedherein. For example, the series of polypeptide chains can be covalentlylinked using a suitable chemical or a disulphide bond. Examples ofnon-covalent bonds include hydrogen bonds, ionic bonds, Van der Waalsforces, and hydrophobic interactions. A non-covalent bond contemplatedby the present disclosure is the interaction between a V_(H) and aV_(L), e.g., in some forms of diabody or a triabody or a tetrabody or aFv.

The term “polypeptide chain” will be understood to mean from theforegoing paragraph to mean a series of contiguous amino acids linked bypeptide bonds.

As the term suggests, “anti-Fn14 antibody” means an antibody thatspecifically binds to Fn14.

Reference herein to antibody CRCBT-06-001 is to an antibody comprising aV_(H) comprising a sequence set forth in SEQ ID NO: 15 and a V_(L)comprising a sequence set forth in SEQ ID NO: 22.

Reference herein to antibody CRCBT-06-002 is to an antibody comprising aV_(H) comprising a sequence set forth in SEQ ID NO: 16 and a V_(L)comprising a sequence set forth in SEQ ID NO: 23.

Reference herein to antibody CRCBT-06-003 is to an antibody comprising aV_(H) comprising a sequence set forth in SEQ ID NO: 17 and a V_(L)comprising a sequence set forth in SEQ ID NO: 24.

Reference herein to antibody CRCBT-06-004 is to an antibody comprising aV_(H) comprising a sequence set forth in SEQ ID NO: 18 and a V_(L)comprising a sequence set forth in SEQ ID NO: 25.

Reference herein to antibody CRCBT-06-005 is to an antibody comprising aV_(H) comprising a sequence set forth in SEQ ID NO: 19 and a V_(L)comprising a sequence set forth in SEQ ID NO: 26.

Reference herein to antibody CRCBT-06-006 is to an antibody comprising aV_(H) comprising a sequence set forth in SEQ ID NO: 20 and a V_(L)comprising a sequence set forth in SEQ ID NO: 27.

Reference herein to antibody CRCBT-06-007 is to an antibody comprising aV_(H) comprising a sequence set forth in SEQ ID NO: 21 and a V_(L)comprising a sequence set forth in SEQ ID NO: 28.

As used herein, “variable region” refers to the portions of the lightand/or heavy chains of an antibody as defined herein that is capable ofspecifically binding to an antigen and includes amino acid sequences ofCDRs; i.e., CDR1, CDR2, and CDR3, and FRs. For example, the variableregion comprises three or four FRs (e.g., FR1, FR2, FR3 and optionallyFR4) together with three CDRs. V_(H) refers to the variable region ofthe heavy chain. V_(L) refers to the variable region of the light chain.

As used herein, the term “complementarity determining regions” (syn.CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues ofan antibody variable region the presence of which are major contributorsto specific antigen binding. Each variable region typically has threeCDR regions identified as CDR1, CDR2 and CDR3. Each complementaritydetermining region may comprise amino acid residues from a“complementarity determining region” as defined by Kabat et al., (1987and/or 1991). For example, in a heavy chain variable region CDRH1 isbetween residues 31-35, CDRH2 is between residues 50-65 and CDRH3 isbetween residues 95-102. In a light chain CDRL1 is between residues24-34, CDRL2 is between residues 50-56 and CDRL3 is between residues89-97. These CDRs can also comprise numerous insertions, e.g., asdescribed in Kabat (1987 and/or 1991). The present disclosure is notlimited to FRs and CDRs as defined by the Kabat numbering system, butincludes all numbering systems, including the canonical numbering systemor of Chothia and Lesk (1987); Chothia et al. (1989); and/or Al-Lazikaniet al., (1997); the numbering system of Honnegher and Plükthun (2001);the IMGT system discussed in Giudicelli et al., (1997); or the EnhancedChothia Numbering Scheme (http://www.bioinfo.org.uk/mdex.html). In oneexample, the CDRs and/or FRs are defined according to the Kabatnumbering system, e.g., as depicted in FIGS. 11A-11D in bold text.Optionally, heavy chain CDR2 according to the Kabat numbering systemdoes not comprise the five C-terminal amino acids listed herein or anyone or more of those amino acids are substituted with anothernaturally-occurring amino acid. In an additional, or alternative,option, light chain CDR1 does not comprise the four N-terminal aminoacids listed herein or any one or more of those amino acids aresubstituted with another naturally-occurring amino acid. In this regard,Padlan et al., 1995 established that the five C-terminal amino acids ofheavy chain CDR2 and/or the four N-terminal amino acids of light chainCDR1 are not generally involved in antigen binding. In one example, theCDRs and/or FRs are defined according to the Chothia numbering system,e.g., as depicted in FIGS. 9A-9D in underlined text.

As used herein, the term “Kabat numbering system” refers to the schemefor numbering antibody variable regions and identifying CDRs(hypervariable regions) as set out in Kabat et al., (1987 and/or 1991).

As used herein, the term “Chothia numbering system” refers to the schemefor numbering antibody variable regions and identifying CDRs (structuralloops) as set out in of Chothia and Lesk (1987) or Al-Lazikani et al.,(1997).

“Framework regions” (hereinafter FR) are those variable region residuesother than the CDR residues.

As used herein, the term “Fv” shall be taken to mean any protein,whether comprised of multiple polypeptides or a single polypeptide, inwhich a V_(L) and a V_(H) associate and form a complex having an antigenbinding domain, i.e., capable of specifically binding to an antigen(e.g., Fn14). The V_(H) and the V_(L) which form the antigen bindingdomain can be in a single polypeptide chain or in different polypeptidechains. Furthermore an Fv of the disclosure (as well as any protein ofthe disclosure) may have multiple antigen binding sites which may or maynot bind the same antigen. This term shall be understood to encompassfragments directly derived from an antibody as well as proteins producedusing recombinant means. In some examples, the V_(H) is not linked to aheavy chain constant domain C_(H)1 and/or the V_(L) is not linked to alight chain constant domain (C_(L)), e.g., a domain antibody. ExemplaryFv containing polypeptides or proteins include a Fab fragment, a Fab′fragment, a F(ab′) fragment, a scFv, a diabody, a triabody, a tetrabodyor higher order complex, or any of the foregoing linked to a constantregion or domain thereof, e.g., C_(H)2 or C_(H)3 domain, e.g., aminibody. A “Fab fragment” consists of a monovalent antigen-bindingfragment of an immunoglobulin, and can be produced by digestion of awhole antibody with the enzyme papain, to yield a fragment consisting ofan intact light chain and a portion of a heavy chain or can be producedusing recombinant means. A “Fab′ fragment” of an antibody can beobtained by treating a whole antibody with pepsin, followed byreduction, to yield a molecule consisting of an intact light chain and aportion of a heavy chain comprising a V_(H) and a single constantdomain. Two Fab′ fragments are obtained per antibody treated in thismanner. A Fab′ fragment can also be produced by recombinant means. An“F(ab′)₂ fragment” of an antibody consists of a dimer of two Fab′fragments held together by two disulfide bonds, and is obtained bytreating a whole antibody molecule with the enzyme pepsin, withoutsubsequent reduction. An “Fab₂” fragment is a recombinant fragmentcomprising two Fab fragments linked using, for example a leucine zipperor a C_(H)3 domain. A “single chain Fv” or “scFv” is a recombinantmolecule containing the variable region fragment (Fv) of an antibody inwhich the variable region of the light chain and the variable region ofthe heavy chain are covalently linked by a suitable, flexiblepolypeptide linker. A discussion of exemplary Fv containing proteinsfalling within the scope of this term is provided herein below.

As used herein, the term “antigen binding domain” shall be taken to meana region of an antibody that is capable of specifically binding to anantigen, i.e., a V_(H) or a V_(L) or a Fv or a variable region asdefined herein. The antigen binding domain need not be in the context ofan entire antibody, e.g., it can be in isolation (e.g., a domainantibody) or in another form, e.g., as described herein, such as a scFv.

The term “constant region” (syn. CR) as used herein, refers to a portionof an antibody comprising at constant domains and which is generally(though not necessarily) glycosylated and which binds to one or more Fcreceptors and/or components of the complement cascade (e.g., conferseffector functions). The heavy chain constant region can be selectedfrom any of the five isotypes: α, δ, ε, γ, or μ. Furthermore, heavychains of various subclasses (such as the IgG subclasses of heavychains) are responsible for different effector functions and thus, bychoosing the desired heavy chain constant region, proteins with desiredeffector function can be produced. Exemplary heavy chain constantregions are gamma 1 (IgG1), gamma 2 (IgG2) and gamma 3 (IgG3).

A “constant domain” is a domain in an antibody the sequence of which ishighly similar in antibodies/antibodies of the same type, e.g., IgG orIgM or IgE. A constant region of an antibody generally comprises aplurality of constant domains, e.g., the constant region of γ, α and δheavy chains comprise three constant domains and the Fc of γ, α and δheavy chains comprise two constant domains. A constant region of μ and εheavy chains comprises four constant domains and the Fc region comprisestwo constant domains.

As used herein, the term “binds” in reference to the interaction of aprotein or an antigen binding domain thereof with an antigen means thatthe interaction is dependent upon the presence of a particular structure(e.g., an antigenic determinant or epitope) on the antigen. For example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody binds to epitope “A”, thepresence of a molecule containing epitope “A” (or free, unlabeled “A”),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled “A” bound to the antibody.

As used herein, the term “specifically binds” shall be taken to mean aprotein of the disclosure reacts or associates more frequently, morerapidly, with greater duration and/or with greater affinity with aparticular antigen or antigens or cell expressing same than it does withalternative antigens or cells. For example, a protein that specificallybinds to an antigen binds that antigen with greater affinity (e.g., 20fold or 40 fold or 60 fold or 80 fold to 100 fold or 150 fold or 200fold greater affinity), avidity, more readily, and/or with greaterduration than it binds to other antigens, e.g., to other TNF superfamilyreceptors or to antigens commonly recognized by polyreactive naturalantibodies (i.e., by naturally occurring antibodies known to bind avariety of antigens naturally found in humans). It is also understood byreading this definition that, for example, a protein that specificallybinds to a first antigen may or may not specifically bind to a secondantigen. As such “specific binding” does not necessarily requireexclusive binding or non-detectable binding of another antigen, this ismeant by the term “selective binding”.

As used herein, reference to a “similar” level of binding will beunderstood to mean that an antibody binds to an antigen at a levelwithin about 30% or 25% or 20% of the level at which it binds to anotherantigen. This term can also mean that one antibody binds to an antigenat a level within about 30% or 25% or 20% of the level at which anotherantibody binds to the same antigen.

As used herein, reference to “substantially the same level” of bindingwill be understood to mean that an antibody binds to an antigen at alevel within about 15% or 10% or 5% of the level at which it binds toanother antigen. This term can also mean that one antibody binds to anantigen at a level within about 5% or 4% or 3% of the level at whichanother antibody binds to the same antigen.

As used herein, the term “epitope” (syn. “antigenic determinant”) shallbe understood to mean a region of Fn14 to which an antibody designatedCRCBT-06-001 and/or CRCBT-06-002 and/or CRCBT-06-003 and/or CRCBT-06-004and/or CRCBT-06-005 and/or CRCBT-06-006 and/or CRCBT-06-007 binds. Thisterm is not necessarily limited to the specific residues or structure towhich the antibody makes contacts. For example, this term includes theregion spanning amino acids contacted by the antibody and/or 5-10 or 2-5or 1-3 amino acids outside of this region. In some example, the epitopeis a series of consecutive amino acids from Fn14. However, an epitopecan also comprise a series of discontinuous amino acids that arepositioned close to one another when Fn14 is folded, i.e., a“conformational epitope”. In this regard, the term “epitope” canencompass a single polypeptide comprising some or all of the series ofdiscontinuous amino acids sufficient to bind to an Fn14-binding proteinof the present disclosure and/or can comprise a series of peptidescomprising the series of amino acids. The skilled artisan will also beaware that the term “epitope” is not limited to peptides orpolypeptides. For example, antibodies are capable of binding tocarbohydrates or glycosylated peptides or polypeptides, phosphates orphospho-peptides or polypeptides amongst other epitopes. An epitope orpeptide or polypeptide comprising same can be administered to an animalto generate antibodies against the epitope.

By “isolated” is meant that the protein is substantially removed fromits naturally-occurring environment, e.g., is in a heterologousenvironment and/or that it is substantially free of contaminatingagents, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or96% or 97% or 98% or 99% free of contaminating agents.

The term “competitively inhibits” shall be understood to mean that aprotein of the disclosure reduces or prevents binding of a recitedantibody produced to Fn14 or a fragment thereof. It will be apparentfrom the foregoing that the protein need not completely inhibit bindingof the antibody, rather it need only reduce binding by a statisticallysignificant amount, for example, by at least about 10% or 20% or 30% or40% or 50% or 60% or 70% or 80% or 90% or 95%. Methods for determiningcompetitive inhibition of binding are known in the art and/or describedherein. For example, the antibody is exposed to Fn14 or a fragmentthereof either in the presence or absence of the protein. If lessantibody binds in the presence of the protein than in the absence of theprotein, the protein is considered to competitively inhibit binding ofthe antibody. In one example, the protein and antibody are exposed toFn14 substantially simultaneously. Additional methods for determiningcompetitive inhibition of binding will be apparent to the skilledartisan and/or described herein. In one example, the antigen bindingdomain of the protein competitively inhibits binding of the antibody.

Reference herein to “an epitope comprising residues contained within” arecited sequence will be understood to mean that the epitope (which canbe conformational) to which a protein binds comprises residues withinthe recited sequence, however may contain additional residues. Theresidues contained within the sequence are sufficient to permit thesequence to bind to a protein described herein according to any example.

By “overlapping” in the context of two epitopes shall be taken to meanthat two epitopes share a sufficient number of amino acid residues topermit an antibody that binds to one epitope to competitively inhibitthe binding of an antibody that binds to the other epitope. For example,the epitopes share at least one or two or three or four or five or sixor seven or eight or nine or ten amino acids.

As used herein, the term “does not detectably bind” shall be understoodto mean that a protein, e.g., an antibody, binds to a candidate antigenat a level less than 10%, or 8% or 6% or 5% above background. Thebackground can be the level of binding signal detected in the absence ofthe protein and/or in the presence of a negative control protein (e.g.,an isotype control antibody) and/or the level of binding detected in thepresence of a negative control antigen. The level of binding is detectedusing biosensor analysis (e.g. Biacore) in which the protein isimmobilized and contacted with an antigen.

As used herein, phrases referring to “reduced binding” or “binding beingat a lower level” in relation to binding of an Fn14-binding protein to apeptide comprising a region of Fn14 or a mutant form thereof will beunderstood to mean that the protein binds to the peptide with anaffinity at least about 2 fold or 5 fold or 10 fold or 20 fold or 40fold or 60 fold less than a control epitope or antigen (e.g. Fn14 or apeptide comprising an amino acid sequence set forth in SEQ ID NO: 2).

As used herein, the term “similar level” in the context of anFn14-binding protein will be understood to mean that a protein of thepresent disclosure binds to two antigens (e.g., Fn14 or a peptidecomprising a sequence set forth in SEQ ID NO: 2 and a peptide comprisinga sequence set forth in any one of SEQ ID NOs: 29-32, 34 or 49-68) withaffinities that are within 2 fold or less of one another, e.g., withinabout 1 fold of one another, such as, within about 0.5 fold of oneanother or the levels of binding are substantially identical.

By “individually” is meant that the disclosure encompasses aFn14-binding protein that binds to the recited antigens or groups ofantigens separately, and that, notwithstanding that individual antigensor groups of antigens may not be separately listed herein theaccompanying claims may define such antigens or groups of antigensseparately and divisibly from each other.

By “collectively” is meant that the invention encompasses any number orcombination of the recited antigens or groups of antigens, and that,notwithstanding that such numbers or combinations of antigens or groupsof antigens may not be specifically listed herein the accompanyingclaims may define such combinations or sub-combinations separately anddivisibly from any other combination of antigens or groups of antigens.

A “Tweak-mediated Fn14 signaling effect” will be understood to mean anyone or group of phenotypes effected by Tweak signaling through Fn14 in acell. Such effects include gene expression changes, NFκB-signaling,induction or inhibition of apoptosis or necrosis, induction orinhibition of angiogenesis or induction or inhibition of cytokinesecretion. Suitable methods for determining Tweak-mediated Fn14signaling effect are described herein.

As used herein, the term “Tweak-induced NFκB-signaling” will beunderstood to mean signal transduction that occurs within a cellexpressing Fn14 that occurs via NFκB as a result of Tweak binding toFn14. Methods for determining such signaling are known in the art and/ordescribed herein. For example, a promoter that comprises a NFκB bindingsite and that facilitates gene expression as a result of NFκB binding isoperably linked to a reporter gene. Tweak-induced NFκB-signaling is thendetermined by detecting expression of the reporter gene, e.g., detectinga fluorescent reporter gene using fluorescence activated cell sorting(FACS). Tweak-induced NFκB-signaling can also be determined by detectingthe level of expression of a gene induced by NFκB, e.g., at the mRNAlevel or protein level.

As used herein, the term “does not detectably induce NFκB-signaling” inthe context of a protein contacted to a cell expressing Fn14 in theabsence of Tweak shall be taken to mean that the protein does not induceNFκB-signaling to a level significantly greater than a control protein,e.g., an isotype control antibody. In some examples, the induction ofsignaling is no more than 1.5 fold or 1.4 fold or 1.3 fold or 1.2 foldor 1.1 fold compared to the induction of signaling by a control protein.Methods for determining NFκB signaling are described herein.

As used herein, the term “neutralize” shall be taken to mean that aprotein is capable of blocking, reducing or preventing any one or moreTweak-mediated signaling effects in a cell through Fn14. Methods fordetermining neutralization are known in the art and/or described herein.

The term “Fn14-mediated condition” shall be taken to encompass anydisease or disorder caused by or associated with excess numbers of cellsexpressing Fn14 and/or overexpression of Fn14 by cells and/or excessactivity of Fn14 and/or an excess level of Tweak, e.g., in serum of asubject. Exemplary Fn14-mediated conditions are cancer, metastasis,excessive vascularization or angiogenesis, autoimmune diseases,inflammatory diseases, neurodegenerative diseases, keloid scarring,graft versus host disease, graft rejection, cardiovascular disease andischemia (including stroke).

As used herein, the term “wasting disorder” refers to a disorder whichinvolves, results at least in part from, or includes loss of weight,muscle atrophy, fatigue, weakness in someone who is not actively tryingto lose weight. Wasting disorders are commonly characterized byinadvertent and/or uncontrolled (in the absence of medical intervention)loss of muscle and/or fat. The term encompasses cachexia or other formsof wasting, e.g., denervation-induced wasting.

The term “wasting disorder associated with another condition” will beunderstood to mean a wasting that is observed in a subject sufferingfrom a condition, i.e., the wasting may result from changes (e.g.,metabolic changes) caused by the condition. In one example, thecondition is an Fn14-mediated condition. In one example, the conditionis caused by or associated with Fn14 expression in a cell (or a cellexpressing Fn14) other than muscle.

As used herein, the term “cachexia” will be understood to refer tometabolic condition associated with an underlying (or another)condition, wherein cachexia is characterized by loss of body weight andloss of muscle with or without loss of fat mass. Cachexia is generallyassociated with increased protein catabolism due to underlyingdisease(s). Contributory factors to the onset of cachexia are anorexiaand metabolic alterations (e.g., increased inflammatory status,increased muscle proteolysis and impaired carbohydrate, protein andlipid metabolism). A prominent clinical feature of cachexia is weightloss in adults (optionally, corrected for fluid retention) or growthfailure in children (excluding endocrine disorders). Anorexia,inflammation, insulin resistance and increased muscle protein breakdownare frequently associated with cachexia. Cachexia is distinct fromstarvation, primary depression, malabsorption and hyperthyroidism and isassociated with increased morbidity. Cachexia can be associated with orresult from (directly or indirectly) various underlying disordersincluding cancer, metabolic acidosis (from decreased protein synthesisand increased protein catabolism), certain infectious diseases (e.g.bacterial infections, including tuberculosis, AIDS), some autoimmunedisorders, addiction to drugs such as amphetamines or cocaine, chronicalcoholism and/or cirrhosis of the liver, chronic inflammatorydisorders, anorexia, neurological conditions and/or neurodegenerativedisease. In one example, cachexia is cancer cachexia (cachexiaassociated with cancer). In other examples, muscle wasting and/orunintended body weight loss associated with neurological conditions,immobility or impaired mobility due to various diseases such asneurodegenerative disease, multiple sclerosis, spinal cord injury, areincluded in the term. Cachexia can be diagnosed based on one or more ofthe following:

-   -   Weight loss of at least 5% over a period of six months (in the        absence of starvation);    -   A BMI<20 together with weight loss; or    -   Appendicular skeletal muscle index consistent with sarcopenia        (males<7.26 kg/m²; females<5.45 kg/m²) together with weight        loss.

As used herein, the term “pre-cachexia” will be understood to mean acondition associated with an underlying condition (e.g., chroniccondition) and characterized by unintentional weight loss of less thanabout 5% of a subject's body weight; and a chronic or recurrent systemicinflammatory response.

As used herein, the term “unintended body weight loss” refers to acondition where the subject is incapable of maintaining a healthy bodyweight or loses a considerable amount of body weight, without actuallyattempting to reduce body weight. For example a body mass index of lessthan 18.5 (or any another BMI range defined by a medical specialist) isconsidered underweight.

For the purposes of the present disclosure, the term “body mass index”is calculated by the following formula: mass (kg)/(height (m)²).

For the purposes of the present disclosure, the term “lean body mass” isto be taken to mean the mass of tissues other than fat (e.g., whiteadipose tissue) in a subject. Lean body mass can be calculated orestimated by estimating a subject's percentage fat content (e.g., usingcalipers) and subtracting this amount from their mass or by using airdisplacement plethysmography or dual energy X-ray absorptiometry (DEXA).

The term “total body mass” will be understood to mean a subject'sweight.

The skilled person will understand from the description herein thatreference to “general health” of a subject includes one or more oflethargy or fatigue, respiratory rate, posture, pain or other scores ofthe health of a subject (e.g. as are known in the art). For example,treatment of a subject with an Fn14-binding protein of the disclosureresulting in improved general health results in one or more of a reducedlevel of lethargy or fatigue, increased respiratory rate, deeperbreathing or improved posture compared to a subject suffering from therelevant condition to who the Fn14-binding protein has not beenadministered.

As used herein, the term “glucose metabolism disorder” shall be taken tomean any disorder in which a subject is unable to or has a reducedability to break down or metabolize or to take up or use one or moreforms of carbohydrate, generally leading to increased levels ofthat/those carbohydrate(s) in the blood stream of the subject. Forexample, the carbohydrate metabolism disorder is associated with orcaused by reduced production by the pancreas of a hormone involved inbreaking down a carbohydrate, e.g., production of amylase. In oneexample, the carbohydrate metabolism disorder is associated with orcaused by reduced production by the pancreas of a hormone involved inuptake of a carbohydrate, e.g., production of insulin. Exemplarycarbohydrate metabolism disorders include Type I diabetes mellitus, TypeII diabetes mellitus, idiopathic Type I diabetes (Type Ib), early-onsetType II diabetes (EOD), youth-onset atypical diabetes (YOAD), maturityonset diabetes of the young (MODY), malnutrition-related diabetes,gestational diabetes, conditions of impaired glucose tolerance (IGT),conditions of impaired fasting plasma glucose, metabolic acidosis,ketosis, syndrome X, hyperglycemia, hypoinsulinemia, insulin resistance,alpha mannosidosis, beta mannosidosis, fructose intolerance,fucosidosis, galactosemia, Leigh disease, mucolipidosis,mucopolysaccharidoses or a complication of any one or more of thepreceding. In one example, the glucose metabolism disorder is diabetes,for example, Type I diabetes.

In one example, a subject suffering from diabetes has a clinicallyaccepted marker of diabetes, such as:

-   -   Fasting plasma glucose of greater than or equal to 7 mmol/L or        126 mg/dl;    -   Casual plasma glucose (taken at any time of the day) of greater        than or equal to 11.1 mmol/L or 200 mg/dl with the symptoms of        diabetes.    -   Oral glucose tolerance test (OGTT) value of greater than or        equal to 11.1 mmol/L or 200 mg/dl measured at a two-hour        interval. The OGTT is given over a two or three-hour time span.

As used herein, the terms “preventing”, “prevent” or “prevention”include administering a therapeutically effective amount of a protein ofthe disclosure sufficient to stop or hinder the development of at leastone symptom of a specified disease or condition.

As used herein, the terms “treating”, “treat” or “treatment” includeadministering a therapeutically effective amount of a protein describedherein sufficient to reduce or eliminate at least one symptom of aspecified disease or condition.

As used herein, the term “subject” shall be taken to mean any animalincluding mammals. Exemplary subjects include humans or non-humanprimates. In one example, the subject is a human.

The term “sample” shall be taken to encompass the recited sample (e.g.,a blood or urine sample) and any fraction thereof (e.g., plasma, serumor buffy coat) or cells derived therefrom (e.g., peripheral bloodmononuclear cells) or processed forms thereof.

The term “ectopically expressed” will be understood to mean that a cellhas been genetically modified to permit it to express the recitedprotein. For example, a cell that ectopically expresses Fn14 has beengenetically modified to permit it to express Fn14. In this regard, priorto genetic modification may have or may not have expressed endogenousFn14.

Antibody-Based Fn14 Binding Proteins

Antibodies

Methods for generating antibodies are known in the art and/or describedin Harlow and Lane (1988) or Zola (1987). Generally, in such methods anFn14 protein or immunogenic fragment or epitope-containing thereof or acell expressing and displaying same (i.e., an immunogen), optionallyformulated with any suitable or desired carrier, adjuvant, orpharmaceutically acceptable excipient, is administered to a non-humananimal subject, for example, a mouse, chicken, rat, rabbit, guinea pig,dog, horse, cow, goat or pig. The immunogen may be administeredintranasally, intramuscularly, sub-cutaneously, intravenously,intradermally, intraperitoneally, or by other known route.

The production of polyclonal antibodies may be monitored by samplingblood of the immunized animal at various points following immunization.One or more further immunizations may be given, if required to achieve adesired antibody titer. The process of boosting and titering is repeateduntil a suitable titer is achieved. When a desired level ofimmunogenicity is obtained, the immunized animal is bled and the serumisolated and stored, and/or the animal is used to generate monoclonalantibodies (mAbs).

Monoclonal antibodies are exemplary antibodies contemplated by thepresent disclosure. The term “monoclonal antibody” or “mAb” or “MAb”refers to a homogeneous antibody population capable of binding to thesame antigen(s) and, for example, to the same epitope within theantigen. This term is not intended to be limited with respect to thesource of the antibody or the manner in which it is made.

For the production of mAbs any one of a number of known techniques maybe used, such as, for example, the procedure exemplified in U.S. Pat.No. 4,196,265 or Harlow and Lane (1988) or Zola (1987).

For example, a suitable animal is immunized with an effective amount ofthe protein or immunogenic fragment or epitope thereof or cellexpressing same under conditions sufficient to stimulate antibodyproducing cells. Rodents such as rabbits, mice and rats are exemplaryanimals, with mice being most commonly used. Mice genetically-engineeredto express human immunoglobulin proteins, and not express murineimmunoglobulin proteins, can also be used to generate an antibody of thepresent disclosure (e.g., as described in WO2002/066630).

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsies of spleens, tonsils or lymph nodes, or from a peripheral bloodsample. The B cells from the immunized animal are then fused with cellsof an immortal myeloma cell, generally derived from the same species asthe animal that was immunized with the immunogen. B cells and immortalcells are fused by incubating a mixture of the cells types in thepresence of an agent or agents (chemical or electrical) that promote thefusion of cell membranes. Fusion methods using Sendai virus have beendescribed by Kohler and Milstein, (1975); and Kohler and Milstein,(1976). Methods using polyethylene glycol (PEG), such as 37% (v/v) PEG,are described by Gefter et al, (1977). The use of electrically inducedfusion methods is also appropriate.

Hybrids are amplified by culture in a selective medium comprising anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary and exemplary agents are aminopterin,methotrexate and azaserine.

The amplified hybridomas are subjected to a functional selection forantibody specificity and/or titer, such as, for example, by flowcytometry and/or immunohistochemstry and/or immunoassay (e.g.radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay,dot immunoassay, and the like). The present disclosure also contemplatessub-cloning of antibody producing cells, e.g., as exemplified herein.

Alternatively, ABL-MYC technology (NeoClone, Madison Wis. 53713, USA) isused to produce cell lines secreting MAbs (e.g., as described in Kumaret al, 1999).

Antibodies can also be produced or isolated by screening a displaylibrary, e.g., a phage display library, e.g., as described in U.S. Pat.No. 6,300,064 EP0368684 and/or U.S. Pat. No. 5,885,793.

Chimeric Antibodies and Proteins

In one example an antibody or Fn14-binding protein of the disclosure isa chimeric antibody or an Fn14-binding protein is a chimeric protein.The term “chimeric proteins” refers to proteins in which an antigenbinding domain V_(H) or V_(L) is from identical with or homologous tocorresponding sequences in proteins derived from a particular species(e.g., murine, such as mouse or rat) or belonging to a particularantibody class or subclass, while the remainder of the chain(s) proteinis identical with or homologous to corresponding sequences in from aproteins derived from another species (e.g., primate, such as human) orbelonging to another antibody class or subclass. In one example, achimeric protein is a chimeric antibody comprising a V_(H) and a V_(L)from a non-human antibody (e.g., a murine antibody) and the remainingregions of the antibody are from a human antibody. The production ofsuch chimeric proteins is known in the art, and may be achieved bystandard means (as described, e.g., in U.S. Pat. No. 6,331,415; U.S.Pat. No. 5,807,715; U.S. Pat. No. 4,816,567 and U.S. Pat. No.4,816,397).

Humanized and Human Antibodies/Proteins

The antibodies or Fn14-binding proteins of the present disclosure may behumanized or human.

The term “humanized protein” shall be understood to refer to a proteincomprising a human-like variable region including CDRs from an antibodyfrom a non-human species grafted onto or inserted into FRs from a humanantibody (this type of antibody is also referred to a “CDR-graftedantibody”). Humanized proteins also include proteins in which one ormore residues of the human protein are modified by one or more aminoacid substitutions and/or one or more FR residues of the human proteinare replaced by corresponding non-human residues. Humanized proteins mayalso comprise residues which are found neither in the human antibody orin the non-human antibody. Any additional regions of the protein (e.g.,Fc region) are generally human. Humanization can be performed using amethod known in the art, e.g., U.S. Pat. No. 5,225,539, U.S. Pat. No.6,054,297, U.S. Pat. No. 7,566,771 or U.S. Pat. No. 5,585,089. The term“humanized protein” also encompasses a super-humanized protein, e.g., asdescribed in U.S. Pat. No. 7,732,578.

In one example, a humanized protein comprises the regions between 27dand 34, 50 and 55, and 89 and 96 in a light chain sequence disclosedherein; and 31 and 35b, 50 and 58, and 95 and 101 in a heavy chainsequence disclosed herein (numbering according to the Kabat numberingsystem). In this regard, Padlan et al., FASEB J., 9: 133-139, 1995presents evidence that these regions are those most likely to bind orcontact antigen.

The term “human protein” as used herein refers to proteins havingvariable and, optionally, constant antibody regions derived from orcorresponding to sequences found in humans, e.g. in the human germlineor somatic cells. The “human” antibodies can include amino acid residuesnot encoded by human sequences, e.g. mutations introduced by random orsite directed mutations in vitro (in particular mutations which involveconservative substitutions or mutations in a small number of residues ofthe protein, e.g. in 1, 2, 3, 4 or 5 of the residues of the protein.These “human antibodies” do not actually need to be generated as aresult of an immune response of a human, rather, they can be generatedusing recombinant means (e.g., screening a phage display library) and/orby a transgenic animal (e.g., a mouse) comprising nucleic acid encodinghuman antibody constant and/or variable regions and/or using guidedselection (e.g., as described in or U.S. Pat. No. 5,565,332). This termalso encompasses affinity matured forms of such antibodies. A humanprotein will also be considered to include a protein comprising FRs froma human antibody or FRs comprising sequences from a consensus sequenceof human FRs and in which one or more of the CDRs are random orsemi-random, e.g., as described in U.S. Pat. No. 6,300,064 and/or U.S.Pat. No. 6,248,516.

Human proteins or antibodies which recognize a selected epitope can alsobe generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al, 1988).

A human Fn14-binding protein of the disclosure comprises a variableregion of a human antibody.

Synhumanized and Primatized Proteins

The Fn14-binding proteins of the present disclosure may be synhumanizedproteins. The term “synhumanized protein” refers to a protein preparedby a method described in WO2007/019620. A synhumanized Fn14-bindingprotein includes a variable region of an antibody, wherein the variableregion comprises FRs from a New World primate antibody variable regionand CDRs from a non-New World primate antibody variable region. Forexample, a synhumanized Fn14-binding protein includes a variable regionof an antibody, wherein the variable region comprises FRs from a NewWorld primate antibody variable region and CDRs from a mouse antibody,e.g., as described herein. In one example, the synhumanized Fn14-bindingprotein is an Fn14-binding antibody in which one or both of the variableregions are synhumanized.

The Fn14-binding proteins of the present disclosure may be primatizedproteins. A “primatized protein” comprises variable region(s) from anantibody generated following immunization of a non-human primate (e.g.,a cynomolgus macaque). Optionally, the variable regions of the non-humanprimate antibody are linked to human constant regions to produce aprimatized antibody. Exemplary methods for producing primatizedantibodies are described in U.S. Pat. No. 6,113,898.

De-Immunized Antibodies and Proteins

The present disclosure also contemplates a de-immunized antibody orFn14-binding protein. De-immunized antibodies and Fn14-binding proteinshave one or more epitopes, e.g., B cell epitopes or T cell epitopesremoved (i.e., mutated) to thereby reduce the likelihood that a subjectwill raise an immune response against the antibody or protein. Methodsfor producing de-immunized antibodies and proteins are known in the artand described, for example, in WO00/34317, WO2004/108158 andWO2004/064724.

Methods for introducing suitable mutations and expressing and assayingthe resulting protein will be apparent to the skilled artisan based onthe description herein.

Antibody Variable Region Containing Proteins

Single-Domain Antibodies

In some examples, an Fn14-binding protein of the disclosure is asingle-domain antibody (which is used interchangeably with the term“domain antibody” or “dAb”). A single-domain antibody is a singlepolypeptide chain comprising all or a portion of the heavy chainvariable region of an antibody. In certain example, a single-domainantibody is a human single-domain antibody (Domantis, Inc., Waltham,Mass.; see, e.g., U.S. Pat. No. 6,248,516; WO90/05144 and/orWO2004/058820).

Diabodies, Triabodies, Tetrabodies

Exemplary Fn14-binding proteins comprising an antibody antigen bindingdomain are diabodies, triabodies, tetrabodies and higher order proteincomplexes such as those described in WO98/044001 and WO94/007921.

For example, a diabody is a protein comprising two associatedpolypeptide chains, each polypeptide chain comprising the structureV_(L)-X-V_(H) or V_(H)-X-V_(L), wherein V_(L) is an antibody light chainvariable region, V_(H) is an antibody heavy chain variable region, X isa linker comprising insufficient residues to permit the V_(H) and V_(L)in a single polypeptide chain to associate (or form an Fv) or is absent,and wherein the V_(H) of one polypeptide chain binds to a V_(L) of theother polypeptide chain to form an antigen binding site, i.e., to forman Fv molecule capable of specifically binding to one or more antigens.The V_(L) and V_(H) can be the same in each polypeptide chain or theV_(L) and V_(H) can be different in each polypeptide chain so as to forma bispecific diabody (i.e., comprising two Fvs having differentspecificity).

Single Chain Fv (scFv) Fragments

The skilled artisan will be aware that scFvs comprise V_(H) and V_(L)regions in a single polypeptide chain. The polypeptide chain furthercomprises a polypeptide linker between the V_(H) and V_(L) which enablesthe scFv to form the desired structure for antigen binding (i.e., forthe V_(H) and V_(L) of the single polypeptide chain to associate withone another to form a Fv). For example, the linker comprises in excessof 12 amino acid residues with (Gly₄Ser)₃ being one of the more favoredlinkers for a scFv.

The present disclosure also contemplates a disulfide stabilized Fv (ordiFv or dsFv), in which a single cysteine residue is introduced into aFR of V_(H) and a FR of V_(L) and the cysteine residues linked by adisulfide bond to yield a stable Fv (see, for example, Brinkmann et al.,1993).

Alternatively, or in addition, the present disclosure provides a dimericscFv, i.e., a protein comprising two scFv molecules linked by anon-covalent or covalent linkage, e.g., by a leucine zipper domain(e.g., derived from Fos or Jun) (see, for example, Kruif and Logtenberg,1996). Alternatively, two scFvs are linked by a peptide linker ofsufficient length to permit both scFvs to form and to bind to anantigen, e.g., as described in US20060263367.

For a review of scFv, see Plückthun (1994).

Minibodies

The skilled artisan will be aware that a minibody comprises the V_(H)and V_(L) domains of an antibody fused to the C_(H)2 and/or C_(H)3domain of an antibody. Optionally, the minibody comprises a hinge regionbetween the V_(H) and a V_(L), sometimes this conformation is referredto as a Flex Minibody. A minibody does not comprise a C_(H)1 or a C_(L).In one example, the V_(H) and V_(L) domains are fused to the hingeregion and the C_(H)3 domain of an antibody. At least one of thevariable regions of said minibody binds to Fn14 in the manner of thedisclosure. Exemplary minibodies and methods for their production aredescribed, for example, in WO94/09817.

Other Antibody Variable Region Containing Proteins

The present disclosure also contemplates other variable regioncontaining Fn14-binding proteins, such as:

-   (i) “key and hole” bispecific proteins as described in U.S. Pat. No.    5,731,168;-   (ii) heteroconjugate proteins, e.g., as described in U.S. Pat. No.    4,676,980;-   (iii) heteroconjugate proteins produced using a chemical    cross-linker, e.g., as described in US4,676,;-   (iv) Fab′-SH fragments, e.g., as described in Shalaby (1992);-   (v) single chain Fab; or-   (vi) Fab₃ (e.g., as described in EP19930302894).-   Non-Antibody Based Antigen Binding Domain Containing Proteins-   Immunoglobulins and Immunoglobulin Fragments

An example of a compound of the present disclosure is a proteincomprising a variable region of an immunoglobulin, such as a T cellreceptor or a heavy chain immunoglobulin (e.g., an IgNAR, a camelidantibody).

The term “immunoglobulin” will be understood to include any antigenbinding protein comprising an immunoglobulin domain. Exemplaryimmunoglobulins are antibodies. Additional proteins encompassed by theterm “immunoglobulin” include domain antibodies, camelid antibodies andantibodies from cartilaginous fish (i.e., immunoglobulin new antigenreceptors (IgNARs)). Generally, camelid antibodies and IgNARs comprise aV_(H), however lack a V_(L) and are often referred to as heavy chainimmunoglobulins. Other “immunoglobulins” include T cell receptors.

Heavy Chain Immunoglobulins

Heavy chain immunoglobulins differ structurally from many other forms ofimmunoglobulin (e.g., antibodies), in so far as they comprise a heavychain, but do not comprise a light chain. Accordingly, theseimmunoglobulins are also referred to as “heavy chain only antibodies”.Heavy chain immunoglobulins are found in, for example, camelids andcartilaginous fish (also called IgNAR).

The variable regions present in naturally occurring heavy chainimmunoglobulins are generally referred to as “V_(HH) domains” in camelidIg and V-NAR in IgNAR, in order to distinguish them from the heavy chainvariable regions that are present in conventional 4-chain antibodies(which are referred to as “V_(H) domains”) and from the light chainvariable regions that are present in conventional 4-chain antibodies(which are referred to as “V_(L) domains”).

Heavy chain immunoglobulins do not require the presence of light chainsto bind with high affinity and with high specificity to a relevantantigen. This means that single domain binding fragments can be derivedfrom heavy chain immunoglobulins, which are easy to express and aregenerally stable and soluble.

A general description of heavy chain immunoglobulins from camelids andthe variable regions thereof and methods for their production and/orisolation and/or use is found inter alia in the following referencesWO94/04678, WO97/49805 and WO 97/49805.

A general description of heavy chain immunoglobulins from cartilaginousfish and the variable regions thereof and methods for their productionand/or isolation and/or use is found inter alia in WO2005/118629.

V-Like Proteins

An example of a Fn14-binding protein of the disclosure is a T-cellreceptor. T cell receptors have two V-domains that combine into astructure similar to the Fv module of an antibody. Novotny et al., ProcNatl Acad Sci USA 88: 8646-8650, 1991 describes how the two V-domains ofthe T-cell receptor (termed alpha and beta) can be fused and expressedas a single chain polypeptide and, further, how to alter surfaceresidues to reduce the hydrophobicity directly analogous to an antibodyscFv. Other publications describing production of single-chain T-cellreceptors or multimeric T cell receptors comprising two V-alpha andV-beta domains include WO1999/045110 or WO2011/107595.

Other non-antibody proteins comprising antigen binding domains includeproteins with V-like domains, which are generally monomeric. Examples ofproteins comprising such V-like domains include CTLA-4, CD28 and ICOS.Further disclosure of proteins comprising such V-like domains isincluded in WO1999/045110.

Adnectins

In one example, a Fn14-binding protein of the disclosure is an adnectin.Adnectins are based on the tenth fibronectin type III (¹⁰Fn3) domain ofhuman fibronectin in which the loop regions are altered to conferantigen binding. For example, three loops at one end of the β-sandwichof the ¹⁰Fn3 domain can be engineered to enable an Adnectin tospecifically recognize an antigen. For further details see US20080139791or WO2005/056764.

Anticalins

In a further example, a Fn14-binding protein of the disclosure is ananticalin. Anticalins are derived from lipocalins, which are a family ofextracellular proteins which transport small hydrophobic molecules suchas steroids, bilins, retinoids and lipids. Lipocalins have a rigidβ-sheet secondary structure with a plurality of loops at the open end ofthe conical structure which can be engineered to bind to an antigen.Such engineered lipocalins are known as anticalins. For furtherdescription of anticalins see U.S. Pat. No. 7,250,297B1 orUS20070224633.

Affibodies

In a further example, a Fn14-binding protein of the disclosure is anaffibody. An affibody is a scaffold derived from the Z domain (antigenbinding domain) of Protein A of Staphylococcus aureus which can beengineered to bind to antigen. The Z domain consists of a three-helicalbundle of approximately 58 amino acids. Libraries have been generated byrandomization of surface residues. For further details see EP1641818.

Avimers

In a further example, a Fn14-binding protein of the disclosure is anAvimer. Avimers are multidomain proteins derived from the A-domainscaffold family. The native domains of approximately 35 amino acidsadopt a defined disulphide bonded structure. Diversity is generated byshuffling of the natural variation exhibited by the family of A-domains.For further details see WO2002088171.

DARPins

In a further example, a Fn14-binding protein of the disclosure is aDesigned Ankyrin Repeat Protein (DARPin). DARPins are derived fromAnkyrin which is a family of proteins that mediate attachment ofintegral membrane proteins to the cytoskeleton. A single ankyrin repeatis a 33 residue motif consisting of two α-helices and a β-turn. They canbe engineered to bind different target antigens by randomizing residuesin the first α-helix and a β-turn of each repeat. Their bindinginterface can be increased by increasing the number of modules (a methodof affinity maturation). For further details see US20040132028.

Other Non-Antibody Polypeptides

Other non-antibody proteins comprising binding domains include thosebased on human γ-crystallin and human ubiquitin (affilins), kunitz typedomains of human protease inhibitors, PDZ-domains of the Ras-bindingprotein AF-6, scorpion toxins (charybdotoxin), C-type lectin domain(tetranectins).

Constant Regions

The present disclosure encompasses Fn14-binding proteins comprising avariable region and a constant region or a domain(s) thereof, e.g., Fc,C_(H)2 and/or C_(H)3 domain. The skilled artisan will be aware of themeaning of the terms constant region and constant domain based on thedisclosure herein and references discussed herein.

Constant region sequences useful for producing the Fn14-binding proteinsof the present disclosure may be obtained from a number of differentsources. In some examples, the constant region or portion thereof of theFn14-binding protein is derived from a human antibody. Moreover, theconstant domain or portion thereof may be derived from any antibodyclass, including IgM, IgG, IgD, IgA and IgE, and any antibody isotype,including IgG1, IgG2, IgG3 and IgG4. In one example, the human isotypeIgG1 is used.

A variety of constant region gene sequences are available in the form ofpublicly accessible deposits or the sequence thereof is available frompublicly available databases. Constant regions can be selected having aparticular effector function (or lacking a particular effector function)or with a particular modification to reduce immunogenicity.

In one example, a protein of the present disclosure has or displays aneffector function that facilitates or enables at least partialdepletion, substantial depletion or elimination of cells expressingFn14. Such an effector function may be enhanced binding affinity to Fcreceptors, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cell mediated phagocytosis (ADCP) and/or complementdependent cytotoxicity (CDC).

In one example, the Fn14-binding protein is capable of inducing anenhanced level of effector function.

In one example, the level of effector function induced by the constantregion is enhanced relative to a wild-type Fc region of an IgG1 antibodyor a wild-type Fc region of an IgG3 antibody.

In another example, the constant region is modified to increase thelevel of effector function it is capable of inducing compared to theconstant region without the modification. Such modifications can be atthe amino acid level and/or the secondary structural level and/or thetertiary structural level and/or to the glycosylation of the Fc region.

The skilled addressee will appreciate that greater effector function maybe manifested in any of a number of ways, for example as a greater levelof effect, a more sustained effect or a faster rate of effect.

Exemplary constant region modifications include amino acidsubstitutions, such as, S239D/1332E, numbered according to the EU indexof Kabat or S239D/A330L/1332E, numbered according to the EU index ofKabat.

Additional amino acid substitutions that increase ability of an Fcregion to induce effector function are known in the art and/ordescribed, for example, in U.S. Pat. No. 6,737,056 or U.S. Pat. No.7,317,091.

In one example, the glycosylation of the constant region is altered toincrease its ability to induce enhanced effector function. In someexamples, Fc regions according to the present disclosure comprise acarbohydrate structure that lacks fucose attached (directly orindirectly) to an Fc region, i.e., the Fc region is “afucosylated”. Suchvariants may have an improved ability to induce ADCC. Methods forproducing afucosylated antibodies include, expressing the Fn14-bindingprotein in a cell line incapable of expressing α-1,6-fucosyltransferase(FUT8) (e.g., as described in Yumane-Ohnuki et al., 2004). Other methodsinclude the use of cell lines which inherently produce antibodiescapable of inducing enhanced effector function (e.g. duck embryonicderived stem cells for the production of viral vaccines, WO2008/129058;Recombinant protein production in avian EBX® cells, WO 2008/142124).

Fn14-binding proteins can also comprise an Fc region capable of inducingenhanced levels of CDC. For example, hybrids of IgG1 and IgG3 produceantibodies having enhanced CDC activity (Natsume et al., 2008).

Methods for determining the ability of an antibody or antigen bindingfragment thereof to induce effector function and known in the art and/ordescribed herein.

In another example, the protein comprises one or more amino acidsubstitutions that increase the half-life of the Fn14-binding protein.For example, the Fn14-binding protein comprises a constant regioncomprising one or more amino acid substitutions that increase theaffinity of the constant region for the neonatal Fc region (FcRn). Forexample, the constant region has increased affinity for FcRn at lowerpH, e.g., about pH 6.0, to facilitate Fc/FcRn binding in an endosome. Inone example, the constant region has increased affinity for FcRn atabout pH 6 compared to its affinity at about pH 7.4, which facilitatesthe re-release of Fc into blood following cellular recycling. Theseamino acid substitutions are useful for extending the half life of aprotein, by reducing clearance from the blood.

Exemplary amino acid substitutions include T250Q and/or M428L or T252A,T254S and T266F or M252Y, S254T and T256E or H433K and N434F accordingto the EU numbering system. Additional or alternative amino acidsubstitutions are described, for example, in US20070135620 or U.S. Pat.No. 7,083,784.

Neutralizing Fn14-binding proteins of the present disclosure cancomprise an IgG4 constant region or a stabilized IgG4 constant region.The term “stabilized IgG4 constant region” will be understood to mean anIgG4 constant region that has been modified to reduce Fab arm exchangeor the propensity to undergo Fab arm exchange or formation of ahalf-antibody or a propensity to form a half antibody. “Fab armexchange” refers to a type of protein modification for human IgG4, inwhich an IgG4 heavy chain and attached light chain (half-molecule) isswapped for a heavy-light chain pair from another IgG4 molecule. Thus,IgG4 molecules may acquire two distinct Fab arms recognizing twodistinct antigens (resulting in bispecific molecules). Fab arm exchangeoccurs naturally in vivo and can be induced in vitro by purified bloodcells or reducing agents such as reduced glutathione. A “half antibody”forms when an IgG4 antibody dissociates to form two molecules eachcontaining a single heavy chain and a single light chain.

In one example, a stabilized IgG4 constant region comprises a proline atposition 241 of the hinge region according to the system of Kabat. Thisposition corresponds to position 228 of the hinge region according tothe EU numbering system. In human IgG4, this residue is generally aserine. Following substitution of the serine for proline, the IgG4 hingeregion comprises a sequence CPPC. In this regard, the skilled personwill be aware that the “hinge region” is a proline-rich portion of anantibody heavy chain constant region that links the Fc and Fab regionsthat confers mobility on the two Fab arms of an antibody. The hingeregion includes cysteine residues which are involved in inter-heavychain disulfide bonds. It is generally defined as stretching from Glu226to Pro243 of human IgG1 according to the numbering system of Kabat.Hinge regions of other IgG isotypes may be aligned with the IgG1sequence by placing the first and last cysteine residues forminginter-heavy chain disulphide (S—S) bonds in the same positions (see forexample WO2010/080538).

Mutant Proteins

The present disclosure provides an Fn14-binding protein having at least80% identity to a sequence of the disclosure and having the samefunctional characteristics described or claimed herein.

In one example, an Fn14-binding protein of the disclosure comprises asequence having at least 80% or 85% or 90% or 91% or 92% or 93% or 94%or 95% or 96% or 97% or 98% or 99% identity to a V_(L) sequencedisclosed herein, provided that the sequence comprises a serine at aposition corresponding to residue 57 of SEQ ID NO: 14. In this regard,the inventors have identified several antibodies sharing at least about92% identity over their entire length.

In another example, an Fn14-binding protein of the disclosure comprisesa sequence having at least 70% or 75% or 80% or 85% or 90% or 91% or 92%or 93% or 94% or 95% or 96% or 97% or 98% or 99% identity to a V_(H) ofthe disclosure described herein, provided that the sequence comprisesone or more of a tryptophan at a position corresponding to position 31of SEQ ID NO: 13, a glutamine at a position corresponding to position 54of SEQ ID NO: 13, an arginine or a serine at a position corresponding toposition 101 of SEQ ID NO: 13, a histidine at a position correspondingto position 107 of SEQ ID NO: 13 or a histidine at a positioncorresponding to position 108 of SEQ ID NO: 13. In this regard, theinventors have identified several antibodies sharing at least about 92%identity over their entire length. The inventors have also identified aseries of HCDR1 according to the Kabat numbering system sharing 60%identity over their entire length. The inventors have also identified aseries of HCDR2 according to the Kabat numbering system sharing 74%identity over their entire length. The inventors have also identified aseries of HCDR3 according to the Kabat numbering system sharing about63% identity over their entire length.

As discussed above, it is also known in the art that the five C-terminalresidues of heavy chain CDR2 can be mutated to conservative ornon-conservative amino acid substitutions (26% of residues) (Padlan etal., 1995). This combined with the variable sites identified by theinventors (five) means up to 52% variation (or at least 48% identity) toa sequence of a CDR2 (according to the Kabat numbering system) as taughtherein.

The present disclosure also provides a nucleic acid encoding theforegoing proteins or nucleic acids that hybridize thereto undermoderate to high stringency conditions.

The present disclosure also encompasses nucleic acids encoding a proteincomprising a sequence set forth in any one of SEQ ID NOs: 13-28, whichdiffers from a sequence exemplified herein as a result of degeneracy ofthe genetic code.

The % identity of a nucleic acid or polypeptide is determined by GAP(Needleman and Wunsch. 1970) analysis (GCG program) with a gap creationpenalty=5, and a gap extension penalty=0.3. The query sequence is atleast 50 residues in length, and the GAP analysis aligns the twosequences over a region of at least 50 residues. For example, the querysequence is at least 100 residues in length and the GAP analysis alignsthe two sequences over a region of at least 100 residues. In oneexample, the two sequences are aligned over their entire length.

The present disclosure contemplates mutant forms of an Fn14-bindingprotein of the disclosure. For example, such a mutant Fn14-bindingprotein comprises one or more conservative amino acid substitutionscompared to a sequence set forth herein. In some examples, theFn14-binding protein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5or 4 or 3 or 2 or 1 conservative amino acid substitutions. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain and/or hydropathicity and/or hydrophilicity.

Families of amino acid residues having similar side chains have beendefined in the art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), β-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Hydropathic indices aredescribed, for example in Kyte and Doolittle (1982) and hydrophylicindices are described in, e.g., U.S. Pat. No. 4,554,101.

The present disclosure also contemplates non-conservative amino acidchanges. For example, of particular interest are substitutions ofcharged amino acids with another charged amino acid and with neutral orpositively charged amino acids. In some examples, the Fn14-bindingprotein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or2 or 1 non-conservative amino acid substitutions.

A mutant form of an Fn14-binding protein described herein according toany example retains the ability to specifically bind to Fn14. Methodsfor determining specific binding to Fn14 are described herein. Forexample, a labeled Fn14-binding protein is brought into contact withimmobilized Fn14. Following washing, bound label is detected. Thelabeled Fn14-binding protein is also brought into contact withimmobilized Fn14 and a related protein or a mutant form of Fn14 or afragment of Fn14 and, following washing, bound label is detected.Detection of label bound to

Fn14 but not to the related or mutant protein or a fragment of Fn14indicates that the mutant Fn14-binding protein retains the ability tospecifically bind to Fn14.

In one example, the mutation(s) occur within a FR of an Fn14-bindingprotein of the disclosure. In another example, the mutation(s) occurwithin a CDR of an Fn14-binding protein of the disclosure.

Affinity Maturation

In a further example, an existing Fn14-binding protein of the disclosureis affinity matured to produce an antibody capable of binding to Fn14with increased affinity. For example, the sequence encoding the V_(L)and/or V_(H) is isolated and the CDR encoding region (e.g., the regionencoding CDR3 of the V_(L) and/or V_(H)) is mutated such that one ormore amino acid substitutions is introduced. The resulting mutantFn14-binding protein is then screened for binding to Fn14, e.g., in acompetitive assay.

The Fn14-binding proteins according to the disclosure may be solublesecreted proteins or may be presented as a fusion protein on the surfaceof a cell, or particle (e.g., a phage or other virus, a ribosome or aspore). Exemplary phage display methods are described, for example, inU.S. Pat. No. 5,821,047; U.S. Pat. No. 6,248,516 and U.S. Pat. No.6,190,908. Phage display particles produced using these methods are thenscreened to identify a displayed Fn14-binding protein having aconformation sufficient for binding to a target antigen e.g., Fn14.

Protein Production

In one example, an Fn14-binding protein of the disclosure is produced byculturing a cell line, e.g., a hybridoma under conditions sufficient toproduce the protein, e.g., as described herein and/or as is known in theart.

Recombinant Expression

In the case of a recombinant protein, nucleic acid encoding same isplaced into one or more expression construct, e.g., expressionvector(s), which is/are then transfected into host cells, such as cellsthat can produce a disulphide bridge or bond, such as E. coli cells,yeast cells, insect cells, or mammalian cells. Exemplary mammalian cellsinclude simian COS cells, Chinese Hamster Ovary (CHO) cells, or myelomacells that do not otherwise produce immunoglobulin protein. Molecularcloning techniques to achieve these ends are known in the art anddescribed, for example in Ausubel or Sambrook. A wide variety of cloningand in vitro amplification methods are suitable for the construction ofrecombinant nucleic acids. Methods of producing recombinant antibodiesare also known in the art. See U.S. Pat. Nos. 4,816,567; 7,923,221 andU.S. Pat. No. 7,022,500.

Following isolation, the nucleic acid encoding a protein of thedisclosure is inserted into an expression construct or replicable vectorfor further cloning (amplification of the DNA) or for expression in acell-free system or in cells. For example, the nucleic acid is operablylinked to a promoter,

As used herein, the term “promoter” is to be taken in its broadestcontext and includes the transcriptional regulatory sequences of agenomic gene, including the TATA box or initiator element, which isrequired for accurate transcription initiation, with or withoutadditional regulatory elements (e.g., upstream activating sequences,transcription factor binding sites, enhancers and silencers) that alterexpression of a nucleic acid, e.g., in response to a developmentaland/or external stimulus, or in a tissue specific manner. In the presentcontext, the term “promoter” is also used to describe a recombinant,synthetic or fusion nucleic acid, or derivative which confers, activatesor enhances the expression of a nucleic acid to which it is operablylinked. Exemplary promoters can contain additional copies of one or morespecific regulatory elements to further enhance expression and/or alterthe spatial expression and/or temporal expression of said nucleic acid.

As used herein, the term “operably linked to” means positioning apromoter relative to a nucleic acid such that expression of the nucleicacid is controlled by the promoter.

Cell free expression systems are also contemplated by the presentdisclosure. For example, a nucleic acid encoding an Fn14-binding proteinof the disclosure is operably linked to a suitable promoter, e.g., a T7promoter, and the resulting expression construct exposed to conditionssufficient for transcription and translation. Typical expression vectorsfor in vitro expression or cell-free expression have been described andinclude, but are not limited to the TNT T7 and TNT T3 systems (Promega),the pEXP1-DEST and pEXP2-DEST vectors (Invitrogen).

Many vectors for expression in cells are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, a sequence encoding Fn14-binding proteinof the present disclosure (e.g., derived from the information providedherein), an enhancer element, a promoter, and a transcriptiontermination sequence. The skilled artisan will be aware of suitablesequences for expression of a protein. For example, exemplary signalsequences include prokaryotic secretion signals (e.g., pelB, alkalinephosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeastsecretion signals (e.g., invertase leader, a factor leader, or acidphosphatase leader) or mammalian secretion signals (e.g., herpes simplexgD signal).

Exemplary promoters include those active in prokaryotes (e.g., phoApromoter, β-lactamase and lactose promoter systems, alkalinephosphatase, a tryptophan (trp) promoter system, and hybrid promoterssuch as the tac promoter).

Exemplary promoters active in mammalian cells include cytomegalovirusimmediate early promoter (CMV-IE), human elongation factor 1-α promoter(EF1), small nuclear RNA promoters (U1a and U1b), α-myosin heavy chainpromoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter(RSV), Adenovirus major late promoter, β-actin promoter; hybridregulatory element comprising a CMV enhancer/β-actin promoter or animmunoglobulin promoter or active fragment thereof. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, AUSTRALIAN CELL BANK CRL 1651); human embryonic kidney line (293or 293 cells subcloned for growth in suspension culture; baby hamsterkidney cells (BHK, AUSTRALIAN CELL BANK CCL 10); or Chinese hamsterovary cells (CHO).

Typical promoters suitable for expression in yeast cells such as forexample a yeast cell selected from the group comprising Pichia pastoris,Saccharomyces cerevisiae and S. pombe, include, but are not limited to,the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1promoter, the PHOS promoter, the nmt promoter, the RPR1 promoter, or theTEF1 promoter.

Means for introducing the isolated nucleic acid molecule or a geneconstruct comprising same into a cell for expression are known to thoseskilled in the art. The technique used for a given cell depends on theknown successful techniques. Means for introducing recombinant DNA intocells include microinjection, transfection mediated by DEAE-dextran,transfection mediated by liposomes such as by using lipofectamine(Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNAuptake, electroporation, viral transduction (e.g., using a lentivirus)and microparticle bombardment such as by using DNA-coated tungsten orgold particles (Agracetus Inc., WI, USA) amongst others.

The host cells used to produce the Fn14-binding protein of thisdisclosure may be cultured in a variety of media, depending on the celltype used. Commercially available media such as Ham's F10 (Sigma),Minimal Essential Medium ((MEM), (Sigma), RPM1-1640 (Sigma), andDulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable forculturing mammalian cells. Media for culturing other cell typesdiscussed herein are known in the art.

Isolation of Proteins

A Fn14-binding protein of the present disclosure is can be isolated orpurified.

Methods for purifying an Fn14-binding protein of the disclosure areknown in the art and/or described herein.

When using recombinant techniques, the Fn14-binding protein of thedisclosure can be produced intracellularly, in the periplasmic space, ordirectly secreted into the medium. If the protein is producedintracellularly, as a first step, the particulate debris, either hostcells or lysed fragments, is removed, for example, by centrifugation orultrafiltration. Where the protein is secreted into the medium,supernatants from such expression systems can be first concentratedusing a commercially available protein concentration filter, forexample, an Amicon or Millipore Pellicon ultrafiltration unit. Aprotease inhibitor such as PMSF may be included in any of the foregoingsteps to inhibit proteolysis and antibiotics may be included to preventthe growth of adventitious contaminants.

The protein prepared from the cells can be purified using, for example,ion exchange, hydroxyapatite chromatography, hydrophobic interactionchromatography, gel electrophoresis, dialysis, affinity chromatography(e.g., protein A affinity chromatography or protein G chromatography),or any combination of the foregoing. These methods are known in the artand described, for example in WO99/57134 or Zola (1997).

The skilled artisan will also be aware that an Fn14-binding protein ofthe disclosure can be modified to include a tag to facilitatepurification or detection, e.g., a poly-histidine tag, e.g., ahexa-histidine tag, or a influenza virus hemagglutinin (HA) tag, or aSimian Virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase(GST) tag. For example, the tag is a hexa-his tag. The resulting proteinis then purified using methods known in the art, such as, affinitypurification. For example, a protein comprising a hexa-his tag ispurified by contacting a sample comprising the protein withnickel-nitrilotriacetic acid (Ni-NTA) that specifically binds a hexa-histag immobilized on a solid or semi-solid support, washing the sample toremove unbound protein, and subsequently eluting the bound protein.Alternatively, or in addition a ligand or antibody that binds to a tagis used in an affinity purification method.

Conjugates

The present disclosure also provides conjugates of Fn14-binding proteinsdescribed herein according to any example. Examples of compounds towhich a protein can be conjugated are selected from the group consistingof a radioisotope, a detectable label, a therapeutic compound, acolloid, a toxin, a nucleic acid, a peptide, a protein, a compound thatincreases the half life of the protein in a subject and mixturesthereof. Exemplary therapeutic agents include, but are not limited to ananti-angiogenic agent, an anti-neovascularization and/or othervascularization agent, an anti-proliferative agent, a pro-apoptoticagent, a chemotherapeutic agent or a therapeutic nucleic acid.

A toxin includes any agent that is detrimental to (e.g., kills) cells.For a description of these classes of drugs which are known in the art,and their mechanisms of action, see Goodman et al., (1990). Additionaltechniques relevant to the preparation of immunoglobulin-immunotoxinconjugates are provided in for instance in U.S. Pat. No. 5,194,594.Exemplary toxins include diphtheria A chain, nonbinding active fragmentsof diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI,PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin,sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin,phenomycin, enomycin and the tricothecenes. See, for example,WO93/21232.

Suitable chemotherapeutic agents for forming immunoconjugates of thepresent disclosure include taxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-de-hydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin, antimetabolites (such as methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine,hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents(such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatinand other platinum derivatives, such as carboplatin), antibiotics (suchas dactinomycin (formerly actinomycin), bleomycin, daunorubicin(formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin,mitoxantrone, plicamycin, anthramycin (AMC)).

In one example, an Fn14-binding protein as described herein according toany example is conjugated or linked to another protein, includinganother Fn14-binding protein of the disclosure or a protein comprisingan antibody variable region, such as an antibody or a protein derivedtherefrom, e.g., as described herein. Other proteins are not excluded.Additional proteins will be apparent to the skilled artisan and include,for example, an immunomodulator or a half-life extending protein or apeptide or other protein that binds to serum albumin amongst others.

Exemplary serum albumin binding peptides or protein are described inUS20060228364 or US20080260757.

A variety of radionuclides are available for the production ofradioconjugated proteins. Examples include, but are not limited to, lowenergy radioactive nuclei (e.g., suitable for diagnostic purposes), suchas ¹³C, ¹⁵N, ²H, ¹²⁵I, ¹²³I, ⁹⁹Tc, ⁴³K, ⁵²Fe, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In and thelike. For example, the radionuclide is a gamma, photon, orpositron-emitting radionuclide with a half-life suitable to permitactivity or detection after the elapsed time between administration andlocalization to the imaging site. The present disclosure alsoencompasses high energy radioactive nuclei (e.g., for therapeuticpurposes), such as ¹²⁵I, ¹³¹I, ¹²³I, ¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga,¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re and ¹⁸⁸Re. These isotopes typically produce highenergy α- or β-particles which have a short path length. Suchradionuclides kill cells to which they are in close proximity, forexample neoplastic cells to which the conjugate has attached or hasentered. They have little or no effect on non-localized cells and areessentially non-immunogenic. Alternatively, high-energy isotopes may begenerated by thermal irradiation of an otherwise stable isotope, forexample as in boron neutron-capture therapy (Guan et al., 1998).

In another example, the protein is conjugated to a “receptor” (such asstreptavidin) for utilization in cell pretargeting wherein the conjugateis administered to the patient, followed by removal of unbound conjugatefrom the circulation using a clearing agent and then administration of a“ligand” (e.g., avidin) that is conjugated to a therapeutic agent (e.g.,a radionucleotide).

The Fn14-binding proteins of the present disclosure can be modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. For example, the moieties suitable forderivatization of the protein are physiologically acceptable polymer,e.g., a water soluble polymer. Such polymers are useful for increasingstability and/or reducing clearance (e.g., by the kidney) and/or forreducing immunogenicity of an Fn14-binding protein of the disclosure.Non-limiting examples of water soluble polymers include, but are notlimited to, polyethylene glycol (PEG), polyvinyl alcohol (PVA), orpropropylene glycol (PPG).

In one example, an Fn14-binding protein as described herein according toany example comprises one or more detectable markers to facilitatedetection and/or isolation. For example, the compound comprises afluorescent label such as, for example, fluorescein (FITC),5,6-carboxymethyl fluorescein, Texas red,nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride,rhodamine, 4′-6-diamidino-2-phenylinodole (DAPI), and the cyanine dyesCy3, Cy3.5, Cy5, Cy5.5 and Cy7, fluorescein(5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine(5,6-tetramethyl rhodamine). The absorption and emission maxima,respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm;568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm;703 nm) and Cy7 (755 nm; 778 nm).

Alternatively, or in addition, the Fn14-binding protein as describedherein according to any example is labeled with, for example, afluorescent semiconductor nanocrystal (as described, for example, inU.S. Pat. No. 6,306,610).

Alternatively, or in addition, the Fn14-binding protein is labeled with,for example, a magnetic or paramagnetic compound, such as, iron, steel,nickel, cobalt, rare earth materials, neodymium-iron-boron,ferrous-chromium-cobalt, nickel-ferrous, cobalt-platinum, or strontiumferrite.

Immobilized Proteins

In one example an Fn14-binding protein is immobilized on a solid orsemi-solid matrix. The term “immobilization” is to be understood toinvolve various methods and techniques to fix proteins onto specificmatrices, e.g. as described in WO99/56126 or WO02/26292. For example,immobilization can serve to stabilize the proteins so that its activityis not reduced or adversely modified by biological, chemical or physicalexposure, especially during storage or in single-batch use.

In the meaning of the disclosure, three basic methods can be used forimmobilization:

Various methods for immobilizing a protein on a matrix are known in theart and include crosslinking, binding to a carrier, retention within asemi-permeable matrix.

Exemplary matrices include porous gels, aluminium oxide, bentonite,agarose, starch, nylon or polyacrylamide.

Assaying Activity of a Protein of the Disclosure

Binding Assays

One form of such an assay is an antigen binding assay, e.g., asdescribed in Scopes (1994). Such a method generally involves labelingthe Fn14-binding protein and contacting it with immobilized antigen or afragment thereof, e.g., a protein comprising an extracellular domain ofFn14 fused to an Fc region of an antibody. Following washing to removenon-specific bound protein, the amount of label and, as a consequence,bound protein is detected. Of course, the Fn14-binding protein can beimmobilized and the antigen labeled. Panning-type assays, e.g., asdescribed or exemplified herein can also be used.

Neutralization Assays

Assays for identifying Fn14-binding proteins that neutralize one or moreFn14-mediated Tweak activities will also be apparent to the skilledperson.

For example, a neutralization assay comprises determining anFn14-binding protein that reduces Tweak-induced NFκB-signaling in a cellexpressing Fn14. In one example, a cell expressing Fn14 is produced orobtained that has incorporated into it, e.g., into its genome, apromoter operably linked to a reporter gene, wherein the promoterinduces expression of the promoter in response to binding by NFκB. Thecell is contacted with Tweak (or Tweak fused to an Fc region of anantibody) in the presence or absence of the Fn14-binding protein. Areduced level of expression of the reporter gene in the presence of theFn14-binding protein compared to the absence of the Fn14-binding proteinis indicative of reduction of Tweak-induced NFκB-signaling in a cellexpressing Fn14. In one example, the cell is also contacted with theFn14-binding protein in the absence of Tweak and the level of reportergene expression determined, which permits identification of anFn14-binding protein that does not have agonist activity. By testingmultiple concentrations of the Fn14-binding protein an IC₅₀ isdetermined, i.e., a concentration at which 50% of the maximum inhibitionof NFκB-signaling occurs.

Another means for testing for reduction of Tweak-induced NFκB-signalingin a cell expressing Fn14 is to test for the level of expression of agene the expression of which is induced by NFκB. Such gene expressionchanges can be measured by nucleic acid-based detection assays (e.g.,quantitative RT-PCR) or protein-based assays (e.g., as describedherein). Exemplary genes induced by NFκB include IL-2, IL-6, TNF, BCL2,VEGF and COX2.

Another method for determining the ability of an Fn14-binding protein ofthe disclosure to neutralize Fn14-mediated Tweak activities is areceptor binding assay. In such a method, an Fn14 or soluble formthereof or cell expressing same is immobilized. Labeled Tweak is thencontacted to the immobilized receptor or cell in the presence or absenceof a test Fn14-binding protein and the amount of bound label detected. Areduction in the amount of bound label in the presence of theFn14-binding protein compared to in the absence of the Fn14-bindingprotein indicates that the Fn14-binding protein reduces or preventsbinding of Tweak to Fn14. By testing multiple concentrations of theFn14-binding protein an IC₅₀ is determined, i.e., a concentration atwhich 50% of the maximum inhibition of Tweak binding to Fn14 occurs.

A further method for determining neutralization of Fn14-mediated Tweakactivities is an ability of an Fn14-binding protein to reduce death ofKym1 cells in the presence of Tweak. Kym1 cells are contacted with Tweak(or Tweak fused to an Fc domain of an antibody) in the presence orabsence of an Fn14-binding protein. A reduced level of cell death (e.g.,assessed by propidium iodide uptake) in the presence of the Fn14-bindingprotein compared to in the absence of the Fn14-binding protein indicatesthat the Fn14-binding protein neutralizes of Fn14-mediated Tweakactivities. In one example, the Kym1 cells are also contacted with theFn14-binding protein in the absence of Tweak and the level of cell deathdetermined, which permits identification of an Fn14-binding protein thatdoes not have agonist activity. By testing multiple concentrations ofthe Fn14-binding protein an IC₅₀ is determined, i.e., a concentration atwhich 50% of the maximum inhibition of Kym1 cell death occurs.

Another method for determining the ability of an Fn14-binding protein ofthe disclosure to neutralize Fn14-mediated Tweak activities is tocontact tumor cells with Tweak in the presence or absence of theFn14-binding protein and to detect secretion of a cytokine, such as,IL-6. A lower level of the cytokine in the presence of the Fn14-bindingprotein compared to in the absence of the Fn14-binding protein indicatesthat the Fn14-binding protein neutralizes Fn14-mediated Tweakactivities. In one example, the cells are also contacted with theFn14-binding protein in the absence of Tweak and the level of cytokinesecretion determined, which permits identification of an Fn14-bindingprotein that does not have agonist activity. By testing multipleconcentrations of the Fn14-binding protein an IC₅₀ is determined, i.e.,a concentration at which 50% of the maximum inhibition of cytokinesecretion occurs.

Other assays for determining inhibition of Fn14-mediated Tweakactivities include inhibition of tube formation by endothelial cells(e.g., HUVECs) and/or inhibition of Tweak-induced IL-8 secretion by A375melanoma cells.

Cytokine Secretion Assays

In another example, the activity of a protein of the disclosure isdetermined by contacting a cell with a protein of the disclosure anddetermining secretion of a cytokine (e.g., IL-8) by the cell. Methodsfor determining IL-8 secretion are known in the art.

Cell Killing Assays

In another example, the ability of an Fn14-binding protein of thedisclosure (e.g., linked to a toxic compound or a constant region) isassessed by determining their ability to induce death of a cell. In thecase of a constant region-linked Fn14-binding protein it is desirable toperform such an assay in the presence of immune effector cells and/orcomplement (e.g., to facilitate ADCC/CDC).

In Vivo Therapeutic Efficacy Assays

A Fn14-binding protein of the disclosure can also be tested in vivo.

For example, an Fn14-binding protein can be tested in an animal model ofa wasting disorder as described herein, e.g., in which a non-humanmammal is administered a tumor cell expressing Fn14 under conditions fora wasting disorder to develop. an Fn14-binding protein of the disclosureis then administered and the effect on the wasting disorder is assessed,e.g., by monitoring body weight changes. A Fn14-binding protein thatreduces or prevents loss of body weight or induces a gain in body weightis selected as a potential therapeutic agent.

A Fn14-binding protein of the disclosure can also be selected on thebasis of its ability to reduce or prevent invasiveness of a tumor cell.For example, a tumor cell is implanted into a subject, e.g., into amuscle, and the subject is administered a test Fn14-binding protein (orfor controls, no Fn14-binding protein is administered). A reduction ininvasion of tissue surrounding the tumor cell (e.g., as assessed usinghistopathology) in the presence of the Fn14-binding protein compared toin the absence of the Fn14-binding protein indicates that theFn14-binding protein reduces or prevent invasiveness of a tumor cell.

A Fn14-binding protein of the disclosure can also be assessed fortherapeutic efficacy by determining its ability to slow or preventdevelopment of a tumor in a xenograft model.

A Fn14-binding protein of the disclosure can also be assessed fortherapeutic efficacy by determining it ability to reduce the amount ofangiogenesis or vasculogenesis in a tumor in a xenograft model.

Therapeutic efficacy can also be assessed in animal models of rheumatoidarthritis e.g., a SKG strain of mouse (Sakaguchi et al.), rat type IIcollagen arthritis model, mouse type II collagen arthritis model; amouse model of GVHD (e.g., as described in Trenado (2002)) or a model ofischemic stroke, e.g., aorta/vena cava occlusion, external necktourniquet or cuff, hemorrhage or hypotension, intracranial hypertensionor common carotid artery occlusion, two-vessel occlusion andhypotension, four-vessel occlusion, unilateral common carotid arteryocclusion (in some species only), endothelin-1-induced constriction ofarteries and veins, middle cerebral artery occlusion, spontaneous braininfarction (in spontaneously hypertensive rats), macrosphereembolization, blood clot embolization or microsphere embolization.

Therapeutic efficacy can also be determined by administration of aFn14-binding protein to a model of diabetes, e.g., type 1 diabetes. Forexample, the test subject is a non-obese diabetic (NOD) mouse (a modelof Type I diabetes) or a mouse or rat to which streptozotocin has beenadministered (models of Type I and/or Type II diabetes).

Competitive Binding Assays

Assays for determining an Fn14-binding protein that competitivelyinhibits binding of an antibody of the disclosure will be apparent tothe skilled artisan. For example, the antibody of the disclosure isconjugated to a detectable label, e.g., a fluorescent label or aradioactive label. The labeled antibody and the test Fn14-bindingprotein are then mixed and contacted with Fn14 or an extracellulardomain thereof fused to an Fc region of an antibody or a peptidecomprising an epitope thereof. The level of labeled antibody is thendetermined and compared to the level determined when the labeledantibody is contacted with the Fn14 or Fn14-Fc fusion or a peptidecomprising an epitope thereof in the absence of the Fn14-bindingprotein. If the level of labeled antibody is reduced in the presence ofthe test Fn14-binding protein compared to the absence of theFn14-binding protein, the Fn14-binding protein competitively inhibitsbinding of the antibody.

Optionally, the test Fn14-binding protein is conjugated to a differentlabel than the antibody. This permits detection of the level of bindingof the test Fn14-binding protein to the protein or epitope.

In another example, the test Fn14-binding protein is permitted to bindto Fn14 or Fn14-Fc fusion or a peptide comprising an epitope thereofprior to contacting the Fn14 or Fn14-Fc fusion or a peptide comprisingan epitope thereof with an antibody described herein. A reduction in theamount of bound antibody in the presence of the Fn14-binding proteincompared to in the absence of the Fn14-binding protein indicates thatthe Fn14-binding protein competitively inhibits binding of the antibodyto Fn14. A reciprocal assay can also be performed using labeledFn14-binding protein and first allowing the antibody to bind to Fn14 orFn14-Fc fusion or a peptide comprising an epitope thereof. In this case,a reduced amount of labeled Fn14-binding protein bound to Fn14 orFn14-Fc fusion or a peptide comprising an epitope thereof in thepresence of the antibody compared to in the absence of antibodyindicates that the Fn14-binding protein competitively inhibits bindingof the antibody to Fn14.

Epitope Mapping Assays

In another example, the epitope bound by an Fn14-binding proteindescribed herein is mapped. Epitope mapping methods will be apparent tothe skilled artisan. For example, a series of overlapping peptidesspanning the Fn14 sequence or a region thereof comprising an epitope ofinterest, e.g., peptides comprising 10-15 amino acids are produced. TheFn14-binding protein is then contacted to each peptide or a combinationthereof and the peptide(s) to which it binds determined. This permitsdetermination of peptide(s) comprising the epitope to which theFn14-binding protein binds. If multiple non-contiguous peptides arebound by the Fn14-binding protein, the Fn14-binding protein may bind aconformational epitope.

In one example, random fragments of Fn14 are expressed on the surface ofphage and the phage contacted with the Fn14-binding protein. Phage boundby the antibody can then be isolated and the amino acid sequence of theexpressed peptide deduced by the encoding nucleic acid contained in thephage. By isolating a series of phage having overlapping peptides apeptide comprising a region of Fn14 comprising residues included in anepitope are identified.

Alternatively, or in addition, amino acid residues within Fn14 aremutated, e.g., by alanine scanning mutagenesis, and mutations thatreduce or prevent Fn14-binding protein binding are determined Anymutation that reduces or prevents binding of the Fn14-binding protein islikely to be within the epitope bound by the Fn14-binding protein.

A further method involves binding Fn14 or a region thereof to animmobilized Fn14-binding protein of the present disclosure and digestingthe resulting complex with proteases. Peptide that remains bound to theimmobilized Fn14-binding protein are then isolated and analyzed, e.g.,using mass spectrometry, to determine their sequence.

A further method involves converting hydrogens in Fn14 or a regionthereof to deutrons and binding the resulting protein to an immobilizedFn14-binding protein of the present disclosure. The deutrons are thenconverted back to hydrogen, the Fn14 or region thereof isolated,digested with enzymes and analyzed, e.g., using mass spectrometry toidentify those regions comprising deutrons, which would have beenprotected from conversion to hydrogen by the binding of an Fn14-bindingprotein described herein.

In the foregoing paragraphs, reference to Fn14 encompasses recombinantFn14, including the extracellular domain thereof.

Affinity Assays

Optionally, the dissociation constant (Kd) or association constant (Ka)or binding constant (K_(D), i.e., Ka/Kd) of an Fn14-binding protein forFn14 or an epitope containing peptide thereof is determined. Theseconstants for an Fn14-binding protein is in one example measured by aradiolabeled or fluorescently-labeled Fn14 binding assay. This assayequilibrates the Fn14-binding protein with a minimal concentration oflabeled Fn14 in the presence of a titration series of unlabeled Fn14.Following washing to remove unbound Fn14, the amount of label isdetermined. According to another example the constants are measured byusing surface plasmon resonance assays, e.g., using BIAcore surfaceplasmon resonance (BIAcore, Inc., Piscataway, N.J.) with immobilizedFn14 or a region thereof.

Pharmaceutical Compositions and Methods of Treatment

Fn14-binding proteins of the disclosure (syn. active ingredients) areuseful for formulations into a pharmaceutical composition forparenteral, topical, oral, or local administration, aerosoladministration, or transdermal administration, for prophylactic or fortherapeutic treatment. The pharmaceutical compositions can beadministered in a variety of unit dosage forms depending upon the methodof administration. For example, unit dosage forms suitable for oraladministration include powder, tablets, pills, capsules and lozenges.

The pharmaceutical compositions of this disclosure are useful forparenteral administration, such as intravenous administration orsubcutaneous administration or administration into a body cavity orlumen of an organ or joint. The compositions for administration willcommonly comprise a solution of the Fn14-binding protein of thedisclosure dissolved in a pharmaceutically acceptable carrier, such asan aqueous carrier. A variety of aqueous carriers can be used, e.g.,buffered saline and the like. The compositions may containpharmaceutically acceptable carriers as required to approximatephysiological conditions such as pH adjusting and buffering agents,toxicity adjusting agents and the like, for example, sodium acetate,sodium chloride, potassium chloride, calcium chloride, sodium lactateand the like. The concentration of Fn14-binding proteins of the presentdisclosure in these formulations can vary widely, and will be selectedprimarily based on fluid volumes, viscosities, body weight and the likein accordance with the particular mode of administration selected andthe patient's needs. Exemplary carriers include water, saline, Ringer'ssolution, dextrose solution, and 5% human serum albumin. Nonaqueousvehicles such as mixed oils and ethyl oleate may also be used. Liposomesmay also be used as carriers. The vehicles may contain minor amounts ofadditives that enhance isotonicity and chemical stability, e.g., buffersand preservatives.

The Fn14-binding protein of the disclosure can be formulated forparenteral administration, e.g., formulated for injection via theintravenous, intramuscular, sub-cutaneous, transdermal, or other suchroutes, including peristaltic administration and direct instillationinto a tumor or disease site (intracavity administration). Thepreparation of an aqueous composition that contains the compounds of thepresent disclosure as an active ingredient will be known to those ofskill in the art.

Suitable pharmaceutical compositions in accordance with the disclosurewill generally include an amount of the Fn14-binding protein of thepresent disclosure admixed with an acceptable pharmaceutical carrier,such as a sterile aqueous solution, to give a range of finalconcentrations, depending on the intended use. The techniques ofpreparation are generally known in the art as exemplified by Remington'sPharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980.

Upon formulation, compounds of the present disclosure will beadministered in a manner compatible with the dosage formulation and insuch amount as is therapeutically/prophylactically effective. Suitabledosages of compounds of the present disclosure will vary depending onthe specific compound, the condition to be treated and/or the subjectbeing treated. It is within the ability of a skilled physician todetermine a suitable dosage, e.g., by commencing with a sub-optimaldosage and incrementally modifying the dosage to determine an optimal oruseful dosage.

Exemplary dosages and timings of administration will be apparent to theskilled artisan based on the disclosure herein.

In some examples, an Fn14-binding protein of the disclosure isadministered with, prior to or after treatment for a condition, e.g.,cancer. Exemplary treatments include radiation therapy, chemotherapy(e.g., caboplatin, siplatin, cyclophosphamide, docetaxal, doxorubicin,erlotinib, etoposide, fluorouracil, irinotecan, methotrexate,paclitaxel, topotecan, vincristine or vinblastine) or administration ofanother drug to treat a condition, e.g., a biologic such as rituximab,trastuzumab, bevacizumab, alemtuzumab, panitumumab, or cetuximab.

In one example, an Fn14-binding protein of the disclosure isadministered with an appetite stimulant (e.g., a melanocortin-4 receptorantagonist, a ghrelin receptor agonist, megestrol acetate or acannabinoid), a drug targeting an inflammatory cytokine (e.g., a TNFantagonist (e.g., etanercept, adalimumab, golimumab, infliximab), ananti-IL-6 antibody (e.g., CNTO-328, ALD-518), a β-adrenoreceptorantagonist, an anabolic steroid, myostatin, an ACE inhibitor oreicosapentaenoic acid).

Diagnostic/Prognostic Assays

It will be apparent from the description herein that the presentdisclosure provides various methods for diagnosing/prognosing conditionsassociated with Fn14 expression.

Protein Detection Assays

One example of the disclosure detects the presence of Fn14 or a cellexpressing same. The amount, level or presence of a protein or cell isdetermined using any of a variety of techniques known to the skilledartisan such as, for example, a technique selected from the groupconsisting of flow cytometry, immunohistochemistry, immunofluorescence,an immunoblot, a Western blot, a dot blot, an enzyme linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay,fluorescence resonance energy transfer (FRET), matrix-assisted laserdesorption/ionization time of flight (MALDI-TOF), electrosprayionization (ESI), mass spectrometry (including tandem mass spectrometry,e.g. LC MS/MS), biosensor technology, evanescent fiber-optics technologyor protein chip technology.

In one example the assay used to determine the amount or level of aprotein is a semi-quantitative assay.

In another example the assay used to determine the amount or level of aprotein is a quantitative assay.

For example, the protein is detected with an immunoassay, e.g., using anassay selected from the group consisting of, immunohistochemistry,immunofluorescence, enzyme linked immunosorbent assay (ELISA),fluorescence linked immunosorbent assay (FLISA), Western blotting,radioimmunoassay (RIA), a biosensor assay, a protein chip assay and animmunostaining assay (e.g. immunofluorescence).

Standard solid-phase ELISA or FLISA formats are particularly useful indetermining the concentration of a protein from a variety of samples.

In one form, an ELISA or FLISA comprises of immobilizing an Fn14-bindingprotein of the disclosure or a protein that binds to a different epitopeof Fn14 on a solid matrix, such as, for example, a membrane, apolystyrene or polycarbonate microwell, a polystyrene or polycarbonatedipstick or a glass support. A sample is then brought into physicalrelation with the immobilized protein, Fn14 is bound or ‘captured’. Thebound Fn14 is then detected using a second labeled compound that bindsto a different epitope of Fn14 (e.g., the Fn14-binding protein of thedisclosure). Alternatively, a third labeled antibody can be used thatbinds the second (detecting) antibody.

It will be apparent to the skilled person that the assay formatsdescribed herein are amenable to high throughput formats, such as, forexample automation of screening processes or a microarray format.Furthermore, variations of the above-described assay will be apparent tothose skilled in the art, such as, for example, a competitive ELISA.

In an alternative example, a polypeptide is detected within or on acell, using methods known in the art, such as, for example,immunohistochemistry or immunofluorescence. Methods usingimmunofluorescence are exemplary, as they are quantitative or at leastsemi-quantitative. Methods of quantitating the degree of fluorescence ofa stained cell are known in the art and described, for example, inCuello, 1984.

Biosensor devices generally employ an electrode surface in combinationwith current or impedance measuring elements to be integrated into adevice in combination with the assay substrate (such as that describedin U.S. Pat. No. 5,567,301). an Fn14-binding protein of the disclosureis incorporated onto the surface of a biosensor device and a biologicalsample contacted to said device. A change in the detected current orimpedance by the biosensor device indicates protein binding to saidFn14-binding protein. Some forms of biosensors known in the art alsorely on surface plasmon resonance to detect protein interactions,whereby a change in the surface plasmon resonance surface of reflectionis indicative of a protein binding to a ligand or antibody (U.S. Pat.No. 5,485,277 and U.S. Pat. No. 5,492,840).

Biosensors are of particular use in high throughput analysis due to theease of adapting such systems to micro- or nano-scales. Furthermore,such systems are conveniently adapted to incorporate several detectionreagents, allowing for multiplexing of diagnostic reagents in a singlebiosensor unit. This permits the simultaneous detection of severalproteins or peptides in a small amount of body fluids.

Imaging Methods

As will be apparent to the skilled artisan from the foregoing, thepresent disclosure also contemplates imaging methods using anFn14-binding protein of the disclosure. For imaging, an Fn14-bindingprotein is generally conjugated to a detectable label, which can be anymolecule or agent that can emit a signal that is detectable by imaging.However, a secondary labeled compound that specifically binds to anFn14-binding protein of the disclosure may also be used. Exemplarydetectable labels include a protein, a radioisotope, a fluorophore, avisible light emitting fluorophore, infrared light emitting fluorophore,a metal, a ferromagnetic substance, an electromagnetic emittingsubstance a substance with a specific magnetic resonance (MR)spectroscopic signature, an X-ray absorbing or reflecting substance, ora sound altering substance.

The Fn14-binding protein of the disclosure (and, if used the labeledsecondary compound) can be administered either systemically or locallyto an organ, or tissue (or tumor, in the case of a cancer) to be imaged,prior to the imaging procedure. Generally, the Fn14-binding protein isadministered in doses effective to achieve the desired optical image ofa tumor, tissue, or organ. Such doses may vary widely, depending uponthe particular Fn14-binding protein employed, condition to be imaged,tissue, or organ subjected to the imaging procedure, the imagingequipment being used, and the like.

In some examples of the disclosure, the Fn14-binding protein is used asin vivo optical imaging agents of tissues and organs in variousbiomedical applications including, but not limited to, imaging oftumors, tomographic imaging of organs, monitoring of organ functions,coronary angiography, fluorescence endoscopy, laser guided surgery,photoacoustic and sonofluorescence methods, and the like.

Examples of imaging methods include magnetic resonance imaging (MRI), MRspectroscopy, radiography, computerized tomography (CT), ultrasound,planar gamma camera imaging, single-photon emission computed tomography(SPECT), positron emission tomography (PET), other nuclearmedicine-based imaging, optical imaging using visible light, opticalimaging using luciferase, optical imaging using a fluorophore, otheroptical imaging, imaging using near infrared light, or imaging usinginfrared light.

In some examples, an imaging agent is tested using an in vitro or invivo assay prior to use in humans, e.g., using a model described herein.

Samples

To the extent that the method of the present disclosure is performed invitro, on an isolated tissue sample, rather than as an in vivo basedscreen, reference to “sample” should be understood as a reference to anysample of biological material derived from an animal such as, but notlimited to, a body fluid (e.g., blood or synovial fluid or cerebrospinalfluid), cellular material (e.g. tissue aspirate), tissue biopsyspecimens or surgical specimens.

The sample which is used according to the method of the presentdisclosure may be used directly or may require some form of treatmentprior to use. For example, a biopsy or surgical sample may requirehomogenization or other form of cellular dispersion prior to use.Furthermore, to the extent that the biological sample is not in liquidform, (if such form is required or desirable) it may require theaddition of a reagent, such as a buffer, to mobilize the sample.

As will be apparent from the preceding description, such an assay mayrequire the use of a suitable control, e.g. a normal or healthyindividual or a typical population, e.g., for quantification.

As used herein, the term “normal individual” shall be taken to mean thatthe subject is selected on the basis that they do not have abnormalnumbers of Fn14 expressing cells or abnormal levels of Tweak.

A “healthy subject” is one that has not been diagnosed as suffering froma condition, e.g., an Fn14-mediated condition and/or is not at risk ofdeveloping the condition.

Alternatively, or in addition, a suitable control sample is a controldata set comprising measurements of the marker being assayed for atypical population of subjects known not to suffer from a condition.

In one example, a reference sample is not included in an assay. Instead,a suitable reference sample is derived from an established data setpreviously generated from a typical population. Data derived fromprocessing, analyzing and/or assaying a test sample is then compared todata obtained for the sample population.

Fn14-Mediated Conditions

The present disclosure encompasses the use of an Fn14-binding protein orantibody or composition described herein to treat any Fn14-mediatedcondition. Exemplary conditions include cancer, metastasis, excessivevascularization or angiogenesis, an autoimmune disease, an inflammatorydisease, a neurodegenerative diseases, keloid scarring, graft versushost disease, graft rejection or ischemia.

In one example, the Fn14-mediated condition is an inflammatory diseaseor an autoimmune disease. In one example, the condition is a connectivetissue disease (including inflammatory arthritis, such as rheumatoidarthritis, psoriatic arthritis, reactive arthritis or gout), lupus(including systemic lupus erythematosus), type 1 diabetes, multiplesclerosis, vasculitis (including Wegener's granulomatosis and HenochSchonlein Syndrome), nephritis (including glomerulonephritis andpneumonitis), atherosclerosis or inflammation of the eye (includinguveitis).

In one example, the autoimmune condition is multiple sclerosis,neuritis, polymyositis, psoriasis, vitiligo, Sjogren's syndrome,arthritis (such as rheumatoid arthritis), Type 1 diabetes, autoimmunepancreatitis, inflammatory bowel diseases, Crohn's disease, ulcerativecolitis, celiac disease, glomerulonephritis, scleroderma, sarcoidosis,autoimmune thyroid diseases, Hashimoto's thyroiditis, Graves disease,myasthenia gravis, Addison's disease, autoimmune uveoretinitis,pemphigus vulgaris, primary biliary cirrhosis, pernicious anemia, orsystemic lupus erythematosis (SLE). In one example, the condition isrheumatoid arthritis or SLE.

In one example, the condition is a connective tissue disease, such asrheumatoid arthritis. In this regard, Dharmapatni et al., (2011) haveshown that Tweak/Fn14 play a role in rheumatoid arthritis.

In one example, the condition is scleroderma (including systemicscleroderma).

In another example, the condition is graft rejection (e.g., allograftrejection) or graft versus host disease (including weight lossassociated with graft versus host disease). In this regard, Tweak/Fn14have been show to play a role in pathogenesis of graft versus hostdisease, e.g., by Zhao et al., (2007).

In another example, the condition is cardiac allograft vasculopathy.

In one example, the condition is graft rejection associated intimalthickening.

In another example the condition is intramyocardial infarction orischemic repurfusion injury. In this regard, Tweak/Fn14 has been shownto play a role in ischemia by, e.g., Frauenknecht et al., (2010) andInta et al., (2008).

In another example, the condition is associated with excessiveangiogenesis and/or neovascularization, e.g., cancer (including solidtumors, leukemias, lymphoma, melanoma, glioma, breast cancer, coloniccancer, gastric cancer, esophageal cancer, renal cell cancer, ovariancancer, cervical cancer, carcinoid cancer, testicular cancer, prostatecancer, head and neck cancer and hepatocellular carcinoma), cancermetastasis, cancer neovascularization, autoimmune disease (includingpsoriasis), nephropathy, retinopathy, preeclampsia, hepatitis, sepsisand macular degeneration.

In one example, the condition is cancer or a metastasis thereof. Theterm “cancer” refers to or describes the physiological condition inmammals that is typically characterized by unregulated cellgrowth/proliferation. Examples of cancer include, but are not limitedto, an adenocarcinoma, a squamous cell carcinoma, adigestive/gastrointestinal cancer, an endocrine cancer, an eye cancer, amusculoskeletal cancer, a breast cancer, a neurologic cancer, agenitourinary cancer, a germ cell cancer, a head and neck cancer, ahematologic/blood cancer, a respiratory cancer, a skin cancer, anAIDS-related malignancy or a gynelogic cancer.

An adenocarcinoma is a cancer of an epithelium that originates inglandular tissue. Exemplary adenocarcinomas include forms of colorectalcancer, lung cancer, cervical cancer, prostate cancer, urachus cancer,vulval cancer, breast cancer, esophageal cancer, pancreatic cancer andgastric cancer.

Digestive/gastrointestinal cancers include anal cancer; bile ductcancer; extrahepatic bile duct cancer; appendix cancer; carcinoid tumor,gastrointestinal cancer; colon cancer; colorectal cancer includingchildhood colorectal cancer; esophageal cancer including childhoodesophageal cancer; gallbladder cancer; gastric (stomach) cancerincluding childhood gastric (stomach) cancer; hepatocellular (liver)cancer including childhood hepatocellular (liver) cancer; pancreaticcancer including childhood pancreatic cancer; sarcoma, rhabdomyosarcoma;rectal cancer; and small intestine cancer.

Endocrine cancers include islet cell carcinoma (endocrine pancreas);adrenocortical carcinoma including childhood adrenocortical carcinoma;gastrointestinal carcinoid tumor; parathyroid cancer; pheochromocytoma;pituitary tumor; thyroid cancer including childhood thyroid cancer;childhood multiple endocrine neoplasia syndrome; and childhood carcinoidtumor.

Eye cancers include intraocular melanoma; and retinoblastoma.

Musculoskeletal cancers include Ewing's family of tumors;osteosarcoma/malignant fibrous histiocytoma of the bone;rhabdomyosarcoma including childhood rhabdomyosarcoma; soft tissuesarcoma including childhood soft tissue sarcoma; clear cell sarcoma oftendon sheaths; and uterine sarcoma.

Neurologic cancers include childhood brain stem glioma; brain tumor;childhood cerebellar astrocytoma; childhood cerebralastrocytoma/malignant glioma; childhood ependymoma; childhoodmedulloblastoma; childhood pineal and supratentorial primitiveneuroectodermal tumors; childhood visual pathway and hypothalamicglioma; other childhood brain cancers; adrenocortical carcinoma; centralnervous system lymphoma, primary; childhood cerebellar astrocytoma;neuroblastoma; craniopharyngioma; spinal cord tumors; central nervoussystem atypical teratoid/rhabdoid tumor; central nervous systemembryonal tumors; and supratentorial primitive neuroectodermal tumorsincluding childhood and pituitary tumor.

Genitourinary cancers include bladder cancer including childhood bladdercancer; renal cell (kidney) cancer; ovarian cancer including childhoodovarian cancer; ovarian epithelial cancer; ovarian low malignantpotential tumor; penile cancer; prostate cancer; renal cell cancerincluding childhood renal cell cancer; renal pelvis and ureter,transitional cell cancer; testicular cancer; urethral cancer; vaginalcancer; vulvar cancer; cervical cancer; Wilms tumor and other childhoodkidney tumors; endometrial cancer; and gestational trophoblastic tumor;

Germ cell cancers include childhood extracranial germ cell tumor;extragonadal germ cell tumor; ovarian germ cell tumor; and testicularcancer.

Head and neck cancers include lip and oral cavity cancer; childhood oralcancer; hypopharyngeal cancer; laryngeal cancer including childhoodlaryngeal cancer; metastatic squamous neck cancer with occult primary;mouth cancer; nasal cavity and paranasal sinus cancer; nasopharyngealcancer including childhood nasopharyngeal cancer; oropharyngeal cancer;parathyroid cancer; pharyngeal cancer; salivary gland cancer includingchildhood salivary gland cancer; throat cancer; and thyroid cancer.

Hematologic/blood cell cancers include leukemia (e.g., acutelymphoblastic leukemia in adults and children; acute myeloid leukemia,e.g., in adults and children; chronic lymphocytic leukemia; chronicmyelogenous leukemia; and hairy cell leukemia); a lymphoma (e.g.,AIDS-related lymphoma; cutaneous T-cell lymphoma; Hodgkin's lymphomaincluding Hodgkin's lymphoma in adults and children; Hodgkin's lymphomaduring pregnancy; non-Hodgkin's lymphoma including non-Hodgkin'slymphoma in adults and children; non-Hodgkin's lymphoma duringpregnancy; mycosis fungoides; Sezary syndrome; Waldenstrom'smacroglobulinemia; and primary central nervous system lymphoma); andother hematologic cancers (e.g., chronic myeloproliferative disorders;multiple myeloma/plasma cell neoplasm; myelodysplastic syndromes; andmyelodysplastic/myeloproliferative disorders).

Respiratory cancers include non-small cell lung cancer; small cell lungcancer; malignant mesothelioma including malignant mesothelioma inadults and children; malignant thymoma; childhood thymoma; thymiccarcinoma; bronchial adenomas/carcinoids including childhood bronchialadenomas/carcinoids; pleuropulmonary blastoma.

Skin cancers include Kaposi's sarcoma; Merkel cell carcinoma; melanoma;basal cell carcinoma and childhood skin cancer.

In a further example, the condition is a wasting disorder, such ascachexia as described in more detail herein. In one example, the wastingdisorder is associated with a condition, such as, cancer, metabolicacidosis, infectious diseases, diabetes, autoimmune immune deficiencysyndrome (AIDS), autoimmune disorders, addiction to drugs, cirrhosis ofthe liver, chronic inflammatory disorders, anorexia, chronic heartfailure, chronic kidney disease, osteoporosis, skeletal muscle disease,motor neuron disease, multiple sclerosis, muscle atrophy andneurodegenerative disease.

In one example, the wasting disorder is cachexia or sarcopenia (e.g.,wasting associated with aging).

In one example, the wasting disorder is cachexia.

In one example, the cachexia is associated with cancer, infectiousdisease (e.g., tuberculosis or leprosy), AIDS, autoimmune disease(including rheumatoid arthritis or type 1 diabetes), cystic fibrosis,drug addiction, alcoholism or liver cirrhosis.

In one example, the cachexia is associated with an autoimmune disease.In one example, the cachexia is associated with rheumatoid arthritis. Inone example, the cachexia is associated with type 1 diabetes.

In one example, the cachexia is associated with cardiac disease.

In one example, the cachexia is associated with chronic kidney disease.

In one example, the cachexia is associated with chronic pulmonaryinflammation.

In one example, the cachexia is associated with instestinalinflammation.

In one example, the cachexia is associated with inflammatory boweldisease.

In one example, the cachexia is associated with aging.

In one example, the cachexia is associated with sepsis.

In one example, the cachexia is associated with AIDS.

In one exemplary form of the present disclosure the wasting disorder iscachexia associated with cancer. Exemplary cancers are described supra.

In one example, the method additionally comprises identifying a subjectsuffering from cachexia. Such a subject can be identified, for example,based on detection of unintentiaonal weight loss following diagnosis ofanother condition (e.g., cancer). For example, the subject can lose atleast 5% of their body weight following diagnosis of another condition(e.g., cancer) or within the previous 30 days.

Kits

The present disclosure also provides therapeutic/prophylactic/diagnostickits comprising compounds of the present disclosure for use in thepresent detection/isolation/diagnostic/prognostic/treatment/prophylacticmethods. Such kits will generally contain, in suitable container means,an Fn14-binding protein of the present disclosure. The kits may alsocontain other compounds, e.g., for detection/isolation/diagnosis/imagingor combined therapy. For example, such kits may contain any one or moreof a range of anti-inflammatory drugs and/or chemotherapeutic orradiotherapeutic drugs; anti-angiogenic agents; anti-tumor cellantibodies; and/or anti-tumor vasculature or anti-tumor stromaimmunotoxins or coaguligands or vaccines.

In one example, the kit is for detecting Fn14 and additionally comprisesa reagent to facilitate detection (a detectable label and/or a substrateof a detectable label. Such kits may additionally comprise a positivecontrol.

In another example, the kit is for isolating a cell or a population ofcells. In such kits an Fn14-binding protein of the disclosure may belabeled with a detectable label to facilitate FACS. The Fn14-bindingprotein may also be labeled with a magnetic or paramagnetic particle tofacilitate MACS. The Fn14-binding protein may also be immobilized on asolid or semi-solid substrate to facilitate isolation.

In a further example, the kit is for treatment or prevention of acondition. In such kits, the Fn14-binding protein may be provided insolution or in a lyophilized form, optionally with a solution forresuspension. The Fn14-binding protein may be conjugated to atherapeutic compound or the kit may include a therapeutic compound forconjugation thereto. As discussed above, the kit may also compriseadditional therapeutic or prophylactic compounds.

Methods of Screening

As will be apparent to the skilled person based on the disclosureherein, the present disclosure provides methods for identifying acompound for the treatment of a wasting disorder. These methods cancomprise additional steps as discussed in the following paragraphs.

The present disclosure also encompasses for the provision of informationconcerning the identified or isolated compound. Accordingly, thescreening methods are further modified by:

-   (i) optionally, determining the structure of the compound; and-   (ii) providing the compound or the name or structure of the compound    such as, for example, in a paper form, machine-readable form, or    computer-readable form.

Naturally, for compound that are known, albeit not previously tested,for their function using a screen provided by the present disclosure,determination of the name and/or structure of the compound is implicit.This is because the skilled artisan will be aware of the name and/orstructure of the compound at the time of performing the screen.

As used herein, the term “providing the compound” shall be taken toinclude any chemical or recombinant synthetic means for producing thecompound or alternatively, the provision of a compound that has beenpreviously synthesized by any person or means. This clearly includesisolating the compound.

In one example, the compound or the name or structure of the compound isprovided with an indication as to its use e.g., as determined by ascreen described herein.

The screening assays can be further modified by:

-   (i) optionally, determining the structure of the compound;-   (ii) optionally, providing the name or structure of the compound    such as, for example, in a paper form, machine-readable form, or    computer-readable form; and-   (iii) providing the compound.

In one example, the synthesized/produced compound or the name orstructure of the compound is provided with an indication as to its usee.g., as determined by a screen described herein.

In one example, the compound is provided in a library of compounds, eachof which or a subset of which may be separated from other members (i.e.,physically isolated). In such cases, a compound is isolated from thelibrary by its identification, which then permits a skilled person toproduce that compound in isolation, e.g., in the absence of othermembers of the library.

The present disclosure includes the following non-limiting examples.

EXAMPLE 1 Generation and Characterization of Monoclonal Antibodies toHuman Fn14

Methods

Production of Tumor Cells Expressing Fn14 and Non-Signaling Fn14

Cloning of Constructs for Tumor Model (Fn14 and Fn14-GPI)

The 4-hydroxytamoxifen (4-OHT) inducible lentiviral infection system(Vince et al., 2007) was used for all constructs. The full lengthwildtype cDNA for human Fn14 (Genbank Accession Number NM_(—)016639) wascloned into the lentiviral vector pF_UAS_Neo at BamH I and Nhe I. Thecontrol construct containing the cDNA for the extracellular region ofhuman Fn14 (Forward CGCGGATCCATGGCTCGGGGCTCGCTGCGC (SEQ ID NO: 37)Reverse GCTGGTGGTCATCCAAAGCAGCCGGAAGGGGGCAGG (SEQ ID NO: 38)) fused tothe TrailR3 GPI anchor coding region (AF020502) was created by overlapPCR (Forward primer CGGCTGCTTTGGATGACCACCAGCCCGGGGACTCCT (SEQ ID NO:39), Reverse primer CGCGCTAGCTTATCAAACAAACACAATCAGAAG (SEQ ID NO: 40))to create Fn14-GPI. Constructs were confirmed by DNA sequencing.

Creation of Cell Lines for In Vivo Tumor Model (Fn14 and Fn14-GPI)

SV40 immortalized mouse embryonic fibroblasts (MEFs) from wildtypeC57BL/6 mice were transformed with human v12Hras by retroviralinfection. v12Hras transformed cells were then infected with lentiviruscarrying DNA constructs for constitutively expressed GEV16 and either4-hydroxytamoxifen inducible hFn14, inducible hFn14-GPI orconstitutively expressing hFn14. Cells were selected with appropriateantibiotics for a minimum of 2 weeks before FACS screening was performedto confirm protein expression. For induction of protein expression,4-OHT was added to normal growth media to a concentration of 100 nM fora minimum of 24 hours. Cells were harvested and live staining wasperformed using commercial antibodies to human Fn14 (Abcam ab21359) andeither an anti-mouse antibody conjugated to R-phycoerythrin (R-PE;Chemicon 1030-09) or AlexaFluor 647 (Invitrogen #A21235). A minimum of10,000 events were recorded for each sample assessed and data wassubsequently analyzed.

Immunization of Mice

A purified recombinant protein comprising Fn14 extracellular domain wasused for immunizations. For generation of anti-Fn14 antibodiesCRCBT-06-001 and CRCBT-06-002, female Balb/c mice were immunizedintraperitoneally (IP) with 15 μg of antigen in PBS emulsified at a 1:1ratio in Complete Freund's Adjuvant (Sigma F 5881) for the primaryimmunization and incomplete Freund's adjuvant (Sigma F 5506) forsubsequent boosts. For generation of anti-Fn14 antibodiesCRCBT-06-005-CRCBT-06-007 mice were immunized with HEK293T cells eithersuspended in PBS and subsequent boosts performed with cells in PBS, orcells were mixed 1:1 in Complete Freund's Adjuvant (Sigma F 5881) forthe primary immunization and incomplete Freund's adjuvant (Sigma F 5506)for subsequent boosts. For all mice, boosts were performed 4 weekly witha final injection given in PBS intraperitoneally 3 days before spleenswere removed and the spleen cells harvested. Test bleeds were taken 5-7days post boost and used for screening for the presence of an immuneresponse to hFn14. Blood collected from mice was allowed to clot at 4°C. overnight. The clot was removed by centrifugation and the serumcollected and stored at −20° C.

Monoclonal Antibody Production

Hybridoma fusions were performed using ClonaCell®-HY Hybridoma Kit#03800, Stemcell Technologies, Australia) essentially according to themanufacturers' instructions. Briefly, the spleen cells were isolated byspleen perfusion before fusion with mouse myeloma SP2/O (1:5 ratio).Selection and cloning steps were performed on methylcellulose-basedsemi-solid in 96 well plates. The culture supernatants were screened byELISA for IgG production and subsequently by flow cytometry forspecificity.

ELISA Screening of Hybridoma Supernatants

Supernatants were screened by ELISA for IgG production. Briefly 96 wellplates were coated overnight at 4° C. with a polyclonal antibody tomouse IgG (Jackson Immuno Research, #715-006-150 Australia) in 0.05%carbonate-Bicarbonate coating buffer followed by blocking with 3% bovineserum albumin (BSA) diluted in phosphate buffer saline (PBS) incubatedfor 1 hour at room temperature (RT). Standard and samples were added(100 μl/well diluted 1:50 in PBS) for 60 min at RT. Bound monoclonalantibody was detected with goat anti mouse IgG-peroxidase (Sigma,#A25554 Australia; 100 μl/well and used at 1/50,000) incubated for 60min at RT followed by incubation with tetramethylbenzidine (TMB)substrate (100 μl/well). Color development was stopped with 2M ofsulphuric acid and the absorbance read at 450 nm.

Flow Cytometry Screening of Hybridoma Supernatants

1×10⁵ MEF v12Hras Fn14+/− (described above) induced with 100 nM 4-OHTwere used as single or mixed cell population (1:1 ratio) prior tostaining. Cells were incubated at 4° C. with samples or controls for 30min in PBS, 2% BSA. Cells were washed and incubated with secondaryantibody (1:100 AlexaFluor 647-conjugated goat anti-mouse IgG,Invitrogen #A21235, Australia) for 30 min at 4° C. in the dark. The BDFACSCanto II was used to perform flow cytometry according to themanufacturer's protocol and data analysis was performed. Experimentswere representative of at least three independent experiments. Purifiedcontrol antibodies include IgG2b (BioLegend #401212), IgG1 (BioLegend#401405), ITEM-1 (BioLegend #314006) and ITEM-2 (eBioscience).

Monoclonal Antibody Purification/Endotoxin Removal and Testing

Purification of Antibody

Approximately 1 liter of serum free conditioned medium was collected perantibody over 4.5 days from a 4× Triple Flask 500 cm² (NUN132913Thermofisher, Aus) of hybridomas cultured in hybridoma serum free media(12045-084, Invitrogen, Aus) supplemented with penicillin-streptomycin(10,000 U/ml). Medium was filtered through a 0.45 μm filter prior topurification. Antibodies were purified by affinity chromatography with a5 ml column of Protein A Sepharose HiTrap MabSelect Xtra (28-4082-60, GEHealthcare, Aus). The Protein A column was equilibrated with buffercontaining 0.02 M sodium phosphate, 250 mM NaCl pH 6.85, and theantibodies were eluted with 0.1 M Glycine/HCl pH 3.0, followed byneutralization with 1 M Tris/HCl pH 9.0. The neutralized eluate wasconcentrated and the buffer was exchanged with PBS using a vivaspin 20column (VS2021, Sartorius, Aus). Endotoxin was removed from samplesusing Detoxi-Gel (20339, Thermo Scientific, Aus) and measured eitherwith E-toxate kits (ET0100, Sigma, Aus) or using the Endosafe®-PTSProtable Test System (5-0.05 Eu/ml range cartridge) and both methodsperformed according to manufacturer's instructions. Endotoxin levels ofall final antibody preparations used for in vivo experiments was belowthe level of detection of the assay (less than 0.05 EU/mg of antibody).

Antibody Isotyping

Antibodies were isotyped using the BD Cytometric Bead Array (CBA) MouseImmunoglobulin Isotyping Kit (Catalogue No. 550026) essentiallyaccording to the manufacturer's instructions.

In Vitro Activity Assays

HEK293T NFκB GFP Assay

HEK293T cells containing a stably integrated NFκB promoter followed byGFP open reading frame were used to test for functionally activeantibodies (Vince et al., 2007). Cells were incubated for 24 hours inthe presence or absence of purified recombinant Tweak-Fc at aconcentration of 5-200 ng/ml. In addition, hybridoma supernatant orcontrols were added to a final dilution of 1:10-1:100. Cells wereharvested and GFP fluorescence was measured by flow cytrometry (BD FACSCanto II, Diva software). A minimum of 10,000 events per sample wererecorded.

As an isotype control an IgG2b anti-Cytochrome C (BD Pharmingen 556433)antibody was used at equivalent concentrations. The hybridomasupernatant from a non-antibody secreting or an irrelevant IgG producinghybridoma was used as a control.

Kym1 Death Assay

Kym1 cell death assays were performed essentially as described in Vinceet al., (2007). Cells were incubated for 24 hours in the presence orabsence of Tweak-Fc, and hybridoma supernatant, purified antibody orcontrols. Total cells were harvested and analyzed by flow cytometry inthe presence of Propidium Iodide (PI; Sigma P4170) at a concentration of50 μg/ml. Data was graphed as the percentage of cell death.

IL-8 Secretion Assay

A375 human melanoma cells were seeded at 1×10⁴ cells per well in a 96well flat bottom plate in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% fetal calf serum and 1% penicillin/streptomycin.Cells were allowed to attach overnight under standard cell cultureconditions of 37° C. with 5% CO₂.

Cells were incubated with either Tweak-Fc (300 ng/ml finalconcentration) or with antibodies alone at the following concentrations:10, 1, 0.1 and 0.01 μg/ml or with both Tweak-Fc and antibody at 300ng/ml and 10 μg/ml final concentration respectively.

After the addition of reagents, cells were incubated for 24 hours understandard tissue culture conditions. Supernatants were collected andcentrifuged at 700 g for 5 minutes at 4° C. Anti-Tweak antibody (MTW-1),Rat IgG1, ITEM-1, IgG2a, IgG2b, IgG1 were purchased from Biolegend, andITEM-4 were purchased from eBioscience.

IL-8 Detection ELISA

After 24 hours, cell culture supernatants were assessed for IL-8 levelsby ELISA (R&D systems) essentially according to the manufacturer'sinstructions. Briefly, the samples and supplied standards were incubatedfor 2 hours at room temperature. After several thorough washes (in thesupplied wash buffer) between incubations, the IL-8 conjugate, followedby substrate solution were added to all samples and incubated at roomtemperature for 60 and 30 minutes, respectively. Absorbance was measuredat 450 nm and 540 nm. The reading at 540 nm was subtracted from the 450reading to account for any plate variability.

DNA Sequencing of Antibody Variable Regions

mRNA was isolated from hybridoma cells using RNeasy midi kit (Qiagen,75144). RT-PCR was done using one-step RT PCR kit (Qiagen, 210212)according to the manufacturer's instructions. Briefly, 1 ng of mRNA wasused as template for cDNA synthesis and variable mouse light and heavychains were amplified with degenerate primers as described in Wang etal, 2000.

The light chain was amplified using 10 pM of 5′-GG GAG CTC GAY ATT GTGMTS ACM CAR WCT MCA-3′ (SEQ ID NO: 41) forward and 5′-GGT GCA TGCF GGATAC AGT TGG TGC AGC ATC-3′ (SEQ ID NO: 42) reverse primer respectively.The heavy chain was amplified using 10 pM of forward primer (MH1: 5′-CTTCCG GAA TTC SAR GTN MAG CTG SAG SAG TC-3′ (SEQ ID NO: 43) or MH2: 5′-CTTCCG GAA TTC SAR GTN MAG CTG SAG SAG TCW GG-3′ (SEQ ID NO: 44)) and thespecific reverse primer (IgG1: 5′-gga aga tct ATA GAC AGA TGG GGG TGTCGT TTT GGC-3′ (SEQ ID NO: 45)). RT-PCR products were separated on 1.5%agarose gels, bands were excised and DNA purified using Wizard®SV Geland PCR Clean-Up System (Promega, A9282). The purified fragments weresub-cloned into pCR® 2.1-TOPO® vector using TOPO TA Cloning® Kit(Invitrogen, K4560-40) and ligations transformed into One Shot® TOP10Electrocomp™ E. coli according to the manufacturer's instructions. X-Galblue-white screening allowed the selection of white colonies that werecultured for DNA isolation using Wizard® Plus SV Minipreps DNAPurification System (Promega, A1460). For each mAb several independentclones were sent for sequencing using universal M13 primers.

To confirm the sequence of CRCBT-06-002 light chain was amplified using10 pM of forward primer MKVP2 5′-ATG GAG WCA GAC ACA CTC CTG YTA TGGT-3′ (SEQ ID NO: 69) and reverse primer 5′-TTT TAT CTC CAG CTT GGT GC-3′(SEQ ID NO: 70; adapted from Debat et al., 2001). RT-PCR products wereseparated on 1.5% agarose gels, bands were excised and DNA purifiedusing Wizard® SV Gel and PCR Clean-Up System (Promega, A9282). Thepurified fragments were sub-cloned into pGEM® vector using pGEM® VectorSystem II (Promega, A3610) and ligations transformed into E. coli JM109competent cells according to the manufacturer's instructions. X-Galblue-white screening allowed the selection of white colonies that werecultured for DNA isolation using Wizard® Plus SV Minipreps DNAPurification System (Promega, A1460). Several independent clones weresent for sequencing using universal primers T7 and SP6

Validation of Antibody CDR/FR Sequences by Mass Spectrometric Analysis

Fab Generation

Fab fragments were generated from CRCBT-06-002 using the ThermoScientific Pierce Mouse IgG1Fab and F(ab′)2 Preparation Kit (cat#44980)according to the manufacturer's instructions with minor modifications.Briefly, 4 mg of antibody were digested by 750 μl of immobilized Ficinin 25 mM cysteine at approximately pH 5.6 for 5 hours at 37° C. withconstant mixing. After incubation, immobilized Ficin resin wascentrifuged at 5,000 g for 1 min and the supernatant was kept on iceImmobilized Ficin resin was washed with 0.5 ml of PBS three times. Thewash fractions were added to the digested antibody and dialysed against20 ml of PBS using a vivaspin20 concentrator 30 KD cut-off (Sartorius,cat# VS15T21) with a diafiltration cup (Sartorius, cat# VSA005) andconcentrated to 0.5 ml. Undigested IgG and Fc fragments were clearedfrom the Fab fragments using protein G Sepharose beads according tomanufacturer's instructions (GE Healthcare, 17-0618-01).

Fab from CRCBT-06-001, CRCBT-06-003, CRCBT-06-004, CRCBT-06-005,CRCBT-06-006 and CRCBT-06-007 were generated using the Thermo ScientificPierce Fab Preparation Kit (cat#44985) according to the manufacturer'sinstructions with minor modifications. Briefly, 5 mg of antibody weredigested by 250 μl of immobilized papain in 20 mM cysteine atapproximately pH 7 for 3 hours at 37° C. with constant mixing. Afterincubation, immobilized papain was centrifuged at 5,000 g for 1 min andthe supernatant was kept on ice. Immobilized papain was washed with 0.5ml of PBS three times. The wash fractions were added to the digestedantibody. Undigested IgG and Fc fragments were cleared from the Fabfragments using protein G Sepharose beads according to manufacturer'sinstructions (GE Healthcare, 17-0618-01).

Sample Preparation for Mass Spectrometric Sequence Analysis

Fab fragments generated from monoclonal antibodies were dissolved in 50mM Tris pH 8.0 and reduced with 10 mM DTT overnight at RT, followed byalkylation with 50 mM IAA at RT in the dark for 30 minutes. Each sampleof Fab was digested with either 0.5 μg of sequence grade Trypsin(Promega) or Chymotrypsin (Promega) at 40° C. for 2 hours. Additionally,each Fab was digested first and then reduced and alkylated. The peptidemixture was separated by RP-HPLC (Dionex, Ultimate 3000) prior to eitherdirect analysis by ESI-microTOF-Q-MS/MS or by spotting onto a MALDI-MStarget plate for subsequent MALDI-tof/tof-MS (both instruments: BrukerDaltonics, Germany) analysis.

The spectra obtained by either instrument were annotated by DataAnalysisand the data transferred to Biotools program (both programs: BrukerDaltonics, Germany). The Mascot search engine (Matrixscience, UK) wereused to search all data against SwissProt database and the constant partand isoform of the antibodies could be confirmed. The sequences of theCDR regions were derived from the DNA sequence and those sequences werematched to the mass spectrometric data using a facility available withthe Biotools program. The mass accuracy applied were a 50 ppm masswindow for MS spectra and 0.8 Da tolerance was used in the MS/MSspectra.

Determining Affinity of Antibodies to Fn14 Using Fn14-Fc

The interaction of mouse anti-Fn14 IgG monoclonal antibodiesCRCBT-06-001 and CRCBT-06-002 with recombinant Fn14-Fc was measuredusing the Bio-Rad ProteOn XPR36 essentially as described by Nahshol etal., (2008). Briefly, a GLM ProteOn sensor chip was activated by flowinga mixture of 0.2 M EDC and 0.05 M sulpho-NHS over the chip (150 μl at 30μl/min for 5 minutes). Rabbit anti-mouse IgG whole molecule (SigmaM-7023) was then coupled to the chip in the vertical orientation byflowing 150 μl of a 50 μg/ml solution in 10 mM acetate buffer (pH 4.5)at 30 μl/min. The remaining coupling sites were then deactivated byflowing 150 μl of 1 M ethanolamine-HCl (pH 8.5) at 30 μl/min. Mouseanti-Fn14 IgG monoclonal antibodies were then bound to the coupledanti-mouse IgG antibody by flowing 150 μl of 100 μg/ml IgG at 25 μl/minover a single channel of the sensor chip in the horizontal direction.Recombinant Fn14-Fc diluted in PBS Tween 20 (0.005%) was then passedover the GLM sensor chip (150 μl, 40 μl/min, contact time 90 seconds anddissociation time of 800 seconds) in the vertical direction in 5channels at the following concentrations: 50 nM, 25 nM, 12.5 nM, 6.25 nMand 3.125 nM. PBS Tween 20 (0.005%) alone was passed over the remainingchannel. Binding sensorgrams were collected and analyzed with theProteOn Manager 2.1 XPR36 software using the Langmuir or bivalentanalyte kinetic models to fit the data and determine the affinityconstant K_(D).

Determining Affinity of Antibodies to Fn14 Using Fn14

The kinetic analysis of the monoclonal antibodies was performed on aProteOn XPR36 system (BioRad) Laboratories) essentially according to theprotocol from Nahshol et al (2008). Monoclonal antibodies against hFn14(50 μg/ml) were covalently coupled to a GMC chip. Analytes were a serialdilution of bacterially expressed and purified Histidine tagged hFn14.The binding sensorgrams were collected, processed and analyzed using theintegrated ProteOn Manager software (BioRad Laboratories). Bindingcurves were fitted using the Langmuir model describing 1:1 bindingstoichiometry.

Characterizing the Nature of the Epitope Bound by CRCBT-06-001

Reduction and Alkylation of Fn14

To determine if the epitope of CRCBT-06-001 is conformational, Fn14-Fcwas reduced with 10 mM Dithiothreitol (DTT) in the presence of 6MGuanidine-HCl pH 8.0 for 45 minutes at 45° C. and alkylated with 50 mMiodoacetamide (IAA) in 1M Tris-HCl pH 8.0 for 1 hour. The protein wasdialyzed extensively into PBS to remove excess DTT and IAA. An ELISA wasperformed by coating the wells of a microtiter plate with the nativeFn14 and R+An Fn14, the anti-Fn14 antibody was allowed to bind, followedby detection with an anti-mouse horse radish peroxidase (HRP) conjugate.The substrate TMB (Sigma) was used and absorbance detected at 450 nmusing a microtiter plate reader. To ensure the integrity of the reducedand alkylated Fn14-Fc, the Fc portion of the fusion protein was detectedusing an anti-Fc-HRP.

Determining Minimal Binding Regions of Fn14 to which the mAb Binds

A phage display approach was used to construct a gene fragment libraryby digesting the human Fn14 gene and expressing the fragments on thesurface of bacteriophage. This is a standard technique which has beenused frequently for epitope mapping of both conformational and linearepitopes.

Preparation and Panning of Human Fn14 Gene Fragment Library

A variation of the method described in Coley et al. 2001 was used toprepare a human Fn14 gene fragment library expressed on M13bacteriophage.

The phagemid vector pHENH6 (Hoogenboom et al., 1991) contains a copy ofthe M13 bacteriophage gene III, coding for the pIII protein on thesurface of phage particles, and a multiple cloning site between theperiplasmic targeting sequence and the functional gene III sequence.This vector was used for expression of hFn14 fragments as a fusion withthe pIII protein. Briefly, oligonucleotide primers flanking the codingregion for Fn14 were used to amplify the entire open reading frame byPCR using hFn14 DNA as template. PCR product was digested with DNase Iand fragments purified and the ends blunted using Vent DNA polymerase.pHENH6 phagemid vector was digested using PstI and subsequently bluntedusing Vent DNA polymerase. The blunted Fn14 gene fragments generated byrandom digest were then ligated into the prepared pHENH6 vector. Ligatedproducts were purified transformed into competent E. coli TG-1 cells byelectroporation. Analysis of the resulting library indicated thatcoverage of the hFn14 sequence was random.

Four rounds of panning were performed by coating 10 wells of amicrotiter plate (NUNC maxisorp) with 2 mg/ml of CRCBT-06-001, the wellswere blocked with 5% skim milk powder in PBS and the library was allowedto bind to the antibody. After 1 hour, the plate was washed to removeunbound phage, and the adherent phage were then eluted with 0.1M glycinepH 2.2 and neutralized. The eluted phage were re-infected in TG1 E. colicells, rescued, amplified and PEG precipitated for the next round ofpanning as described in Coley et al. 2001. Clones from round 4 were DNAsequenced in order to establish the identity of the Fn14 fragments thatbound to CRCBT-06-001 antibody.

Preparation of Gene Fragments Expressed on Phage

The solution structure of Fn14 has recently been described by He et al.,2009, in addition Brown et al., 2006 identified critical residues forTweak ligand interaction. The extracellular domain of Fn14 contains 6cysteine residues and the disulphide pairing was described in He et al.(2009). Fragments of Fn14 as depicted in FIG. 13 were produced,expressed on phage and used to characterize the epitope bound byCRCBT-06-001.

Cloning of hFn14 Constructs for Display on M13 Phage

Oligonucleotide primers, engineered with XhoI and NotI restriction siteswere designed, synthesized (Geneworks) and used to amplify thefull-length extracellular domain and sub-domains 1, 2 and 3 of Homosapiens Fn14 coding sequence by PCR, the products of which weresubsequently A-tailed and ligated into the pGEMT vector (Promega).Plasmid from a single hFn14 extracellular domain/SD1/2/3-pGEMT clone wasprepared and inserts were digested from the vector backbone using theXhoI and NotI restriction enzymes. Restricted inserts were then purifiedand ligated into the phagemid vector pHEN-H6 (Hoogenboom et al., 1991).For hFn14 mutants, mutagenesis was achieved using the PhusionSite-Directed Mutagenesis kit (Finnzymes) and the hFn14 extracellulardomain-pHEN-H6 plasmid as template.

Epitope Mapping Using Phage Expressed Fn14—Method 1

Expressing and Purifying Phage

Glycerol stocks of TG-1 E. coli transformed with the phagemid constructswere used to inoculate 10 mL of YT media containing Ampicillin. Startercultures were grown to log phase before M13K07 helper phage were addedto infect the cells. Helper phage-infected cultures were thentransferred to 200 ml of SB media containing Ampicillin and Kanamycin.Cultures were grown overnight at 30° C. E. coli were subsequentlycollected through centrifugation and discarded. PEG/NaCl solution wasadded to the culture supernatant, precipitating the phage, which weresubsequently harvested through centrifugation, resuspended in phosphatebuffered saline solution and stored at −80° C.

Enzyme Linked Immunosorbent Assays to Determine Binding of Phage toAntibodies

The wells of Maxisorp immunoplates (Nunc) were coated overnight at 4° C.with the target monoclonal antibodies or recombinant Tweak-Fc at aconcentration of 2 μg/mL in PBS. MAb 5G8 was used as a negative controlfor ELISA plates coated with tested antibodies. Recombinant Fc alone wasused as a negative control for ELISA plates coated with recombinantTweak-Fc. Wells of the coated ELISA plates were subsequently blocked for2 hrs with 10% skim milk solution in PBS. 10-fold serial dilutions ofphage were made using 5% skim milk/PBS solution in 2 ml-well microtiterplates and phage dilutions were added to the coated, blocked ELISAplates for 1 hr, with vigorous shaking at room temperature. Phage weresubsequently removed from the wells and the ELISA plates were washed 4×with PBS/0.05% Tween 20. HRP-conjugated anti-M13-phage polyclonal IgGdiluted in PBS was then added to the wells of the ELISA plates for 1 hr,with vigorous shaking at room temperature. The antibody solution wasthen removed from the wells and the ELISA plates were washed 5× withPBS/0.05% Tween 20. ELISAs were developed using TMB substrate and thereaction was stopped using 2 M H₂SO₄ solution. Optical density at 450 nmwas quantified using a Spectramax absorbance spectrophotometer.

Epitope Mapping Using Phage Expressed Fn14—Method 2

Expressing and Purifying Phage

Single colonies of TG-1 E. coli transformed with the phagemid constructswere used to inoculate 10 ml of 2YT media containing Ampicillin (100μg/ml). Starter cultures were grown to log phase before M13K07 helperphage was added to rescue the phage infected E. coli. Helperphage-infected cultures were amplified in 200 ml of 2YT media containingAmpicillin and Kanamycin (50 μg/ml). Cultures were grown overnight at30° C. in a shaking incubator. The culture was centrifuged and the phage(supernatant fraction) was harvested by PEG/NaCl precipitation. Thephage were pelleted by centrifugation and resuspended in PBS and storedat −80° C.

Enzyme Linked Immunosorbent Assays to Measure Reactivity of mAbs withFn14 Mutants

The wells of Maxisorp immunoplates (Nunc) were coated overnight at 4° C.with the target monoclonal antibodies or recombinant Tweak-Fc at aconcentration of 1.5 μg/ml in PBS. mAb 5G8 was used as a negativecontrol for ELISA plates coated with tested antibodies. Wells of thecoated ELISA plates were subsequently blocked for 2 hours with 5% skimmilk solution in PBS. 10-fold serial dilutions of phage were made usingPBS solution and phage dilutions were added to the coated, blocked ELISAplates for 1 hour, shaking at room temperature. Phage was subsequentlyremoved from the wells and the ELISA plates were washed three times withPBS/0.05% Tween 20.

Detection of Phage

Two methods were used for detecting binding of phage mutants.

Method A: HRP-conjugated anti-M13-phage antibody (GE Healthcare) at a1/5000 dilution in PBS was then added to the wells of the ELISA platesfor 1 hour, shaking at room temperature. The antibody solution was thenremoved from the wells and the ELISA plates were washed four times withPBS/0.05% Tween 20. Method B: Biotin-conjugated anti-M13-phage antibody(GE Healthcare) diluted in PBS was then added to the wells of the ELISAplates for 1 hour shaking at room temperature. The antibody solution wasthen removed from the wells and the ELISA plates were washed three timeswith PBS/0.05% Tween 20. HRP-conjugated Streptavidin diluted in PBS wasthen added to the wells of the ELISA plates for 1 hour, shaking at roomtemperature. The solution was then removed from the wells and the ELISAplates were washed three times with PBS/0.05% Tween 20. ELISAs weredeveloped using TMB substrate and the reaction was stopped using 2 MH₂SO₄ solution. Optical density at 450 nm was quantified using aSpectramax absorbance spectrophotometer.

Epitope Mapping of Antibodies Using Synthetic Peptides

Peptides representing sub-domains 1p, 2 and 3 of hFn14 (FIG. 20, PanelsB and C and FIG. 14, Panel A) and for disulfide pair mutants (Cys3&6ΔSand Cys4&5ΔS; FIG. 20 Panels D and E) were synthesized by GLBiochem Ltd.(Shanghai, China) and the cysteine residues for disulfide bonds werechosen as described in the solution structure by He et al., (2009). Anadditional lysine residue was added to the C-terminus in order to attacha biotin moiety and to enable the peptides to bind to neutravidin coatedplates (Pre-blocked; Pierce). For these experiments, a control peptidewith a biotin at the C-terminus was used as a negative control andisotype control antibodies were also used.

The ELISA was performed essentially according to the manufacturer'srecommended protocol (Pierce). Briefly, peptide was coated onto wells(in duplicate) at 1-10 μg/ml for 2 hours at room temperature. Wells werewashed in PBS/0.05% Tween 20/0.1% BSA 3 times and dilutions of eachantibody were added in wash buffer for 1 hour with gentle agitation.After another three washes anti-mouse HRP-conjugated to horse radishperoxidase (HRP; Chemicon) was added at 1/1000 dilution for 1 hourshaking. Finally, after 4 further washes the plate was developed usingTMB (3,3′,5,5′-tetramethylbenzidine) substrate (Pierce), and thereaction was stopped using 2M H₂SO₄ solution. Optical density at 450 nMwas quantified using a Spectromax absorbance spectrophotometer.

Epitope Mapping of Antibodies Using Recombinant Protein

Cloning of hFn14 and mFn14 Constructs for Bacterial Expression

Oligonucleotide primers, engineered with Nde I and Bam HI restrictionsites, were designed, synthesized (Geneworks) and used to amplify thefull-length extracellular domain of the H. sapiens Fn14 (hFn14), themouse Fn14 (mFn14) extracellular domain and mutants of hFn14 (R56P,R56A, R56K, R58K) by PCR. Subsequent products were ligated into thevector pGEM-4Z (Promega) excised with Sma I. Plasmids from single clonewere prepared and inserts were digested from the vector. Restrictedinserts were then purified and inserted into pET-15b (Novagen) forexpression in E. coli.

Expression and Purification of (His)₆-Proteins

pET-15b derived vectors were transformed into Shuffle T7 Express E. coli(New England BioLabs). Expression was induced by 1 mM IPTG at 30° C. forfour hours. Bacterial lysate was loaded onto a 1 mlNi²⁺-nitrilotriacetic acid (Ni²⁺-NTA)-agarose (Qiagen) columnequilibrated with lysis buffer (20 mM Tris-HCl [pH 7.8], 300 mM NaCl,20% [v/v] glycerol, 18 mM imidazole, 1 mM PMSF, Boehringer completeprotease inhibitors). The column was first washed with 20 ml of lysisbuffer and recombinant proteins were eluted by 300 mM imidazole.Recombinant proteins were dialyzed and concentrated to 1 mg/ml in 25 mMHEPES-KOH (pH 7.4), 100 mM NaCl by a Centricon Centrifugal FilterDevices YM-10 (Millipore).

Analytical Gel Filtration Chromatography

(His)₆-proteins were further purified by gel filtration. Protein samples(1 ml) were loaded onto a HiLoad 16/60 Superdex 75 pg column (GEHealthcare) equilibrated with 25 mM HEPES-KOH (pH 7.4), 100 mM NaCl atroom temperature and were chromatographed at a flow rate of 1.5 ml/minon an ÄKTAxpress system (GE Healthcare). Elution profiles were detectedat 280 nm and 1.5 ml fraction were collected.

ELISA Screening of Monoclonal Antibodies with Purified (His)₆-Proteins

The wells of Maxisorp immunoplates (Nunc) were coated overnight at 4° C.with 2 fold serial dilutions of purified histidine tagged proteins (0.5mg/ml to 4 ng/ml). Nonspecific protein binding sites were blocked with1% milk in phosphate-buffered saline (PBS) for 1 hour at 37° C. Plateswere washed with PBST (PBS [10 mM Na-phosphate and 150 mM NaCl], 0.05%Tween 20). Monoclonal antibodies diluted to 1 μg/ml in PBST (PBS 0.005%Tween 20, pH 7.4)+0.1% milk were added and incubated at 37° C. for 2hours in the coated plates. After washing with PBST, a 1:15,000 dilutionof peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L) (Sigma)containing 0.1% milk was added for 1 hour at 37° C. ELISAs weredeveloped using TMB substrate and the reaction was stopped using 2 MH₂SO₄ solution. Optical density at 450 nm was quantified using aSpectramax absorbance spectrophotometer.

Results

Generating a Fn14 Specific Immune Response in Mice Using RecombinantProtein

Mice were immunized using a recombinant protein comprising Fn14extracellular domain in the presence or absence of adjuvant and aftersubsequent boosts a small bleed was taken and sera were isolated. Serumsamples were screened for the presence of IgG antibodies specific tohFn14 by flow cytometry on live hFn14 inducible mouse embryonicfibroblasts (MEFs; FIG. 1). A number of mice displayed a good immuneresponse to hFn14 as indicated by an increase in fluorescence on inducedcells similar to that of the positive control when compared to stainingof non-induced cells (panel B).

To confirm this result, sera were also screened on the human glioma cellline D645 that naturally express Fn14 when cultured in the presence ofserum (FIG. 2). The same mice that tested positive on 4-OHT induced MEFcells also displayed positive staining on D645 cells, with the overallshift in fluorescence comparable to that for the positive control (panelA.iii. compared to panel B) with very minimal background staining seenwhen cells were stained with serum from mice immunized with anon-related antigen (panel A, vi. & vii).

Identifying Mice Producing Antagonistic Anti-Fn14 Antibodies

The serum from immunized mice was assayed for the presence offunctionally active antibodies using two assays, the cell-based reporterassay for NFκB activity and the Kym1 cell death.

Results of the cell-based reporter assay for NFκB activity showed thatexogenous Tweak-Fc induced a strong NFκB activation and that NFκBactivation could be blocked by soluble TweakR or sera from mice thatwere positive for the presence of anti-hFn14 antibodies. In contrast,sera from mice immunized with a non-related antigen or that did not testpositive for anti-hFn14 antibodies did not block NFκB activation.

As the serum from some mice was capable of blocking of NFκB activation,the capability of these antibodies to block Tweak induced cell death wasnext investigated. Results of this assay showed that culturing Kym1cells in the presence of Tweak-Fc led to a dramatic reduction in cellsurvival, and that this effect could be blocked with soluble TweakR orserum from the hFn14 immunized mice that reduced Tweak induced NFκBactivation, confirming the presence of antagonistic anti-hFn14antibodies.

Monoclonal Antibody Production

The spleens from mice displaying the presence of human Fn14 specificantibodies and the capability of blocking Tweak were used for monoclonalantibody production. Hybridoma fusions were performed and subsequentclones were initially screened by ELISA for IgG secretion. Thesupernatants from those clones producing IgG were further assessed bystaining live cells (+/− hFn14; FIG. 3). A large number of clones weregenerated that specifically bound to hFn14 positive cells but not thoselacking hFn14. Panel B is representative of a number of the clonesscreened with no Fn14 binding, moderate or high binding displayed. PanelC displays four antibodies designated CRCBT-06-001, CRCBT-06-002,CRCBT-06-003 and CRCBT-06-004, which clearly display a high level ofbinding to cell surface expressed hFn14.

NFκB Assay to Assess Monoclonal Antibodies for Functional Activity

The supernatants from antibodies CRCBT-06-001 and CRCBT-06-002 were thenassessed for functional activity in the NFκB assay described above.Substantially increased GFP fluorescence is evident in the presence ofsoluble Tweak-Fc but not in the presence of supernatants from either anon-IgG secreting cell line or that secreting a non-related control IgG(FIG. 4, panel A). In the presence of CRCBT-06-001 and CRCBT-06-002containing supernatants, a strong inhibition of NFκB activation isevident, whereas in the presence of other supernatants no visible effecton activation can be seen (panel B).

Given the specificity and blocking capabilities of CRCBT-06-001 andCRCBT-06-002 these hybridomas producing the antibodies were chosen forfurther characterization. Each hybridoma cell line was cloned at leastonce and subsequent cell lines chosen. All sub-clones chosen displayedthe same binding and functional characteristics of the parent celllines.

Isotyping of monoclonal antibodies was performed. AntibodiesCRCBT-06-001 and CRCBT-06-002 were found to be IgG2bκ and IgG1,respectively. CRCBT-06-003 and CRCBT-06-004 were both found to beIgG2bκ.

Monoclonal antibody CRCBT-06-001 was purified and assessed by massspectrometry to ensure a high level of purity. Purified CRCBT-06-001 wasthen assayed for the level of blocking detectable in the NFκB assay(FIG. 4, panel C). At 50 ng/ml and above, CRCBT-06-001 is able to blockthe action of 5 or 50 ng/ml of Tweak-Fc on NFκB activation. The completeblocking of Tweak by CRCBT-06-001 can be seen at a concentration of 1μg/ml. A minor level of activation of Fn14 is seen with higherconcentrations of CRCBT-06-001, however this was not consistently seen.A purified IgG2b isotype control however showed no effect on the actionof Tweak when added to 1 μg/ml (panel A, bottom series).

Transiently Transfected HEK293T Cells for the Immunization of Mice

To generate additional hFn14 antibodies, a cell based immunizationstrategy was used. The transient transfection of HEK293T with an hFn14over-expression construct was optimized. Mice were immunized withtransiently transfected HEK293T cells in the presence or absence ofadjuvant. After 2 subsequent booster immunizations, a small bleed wastaken for screening. Serum from these mice was used to stain MEF cells(+/−hFn14) for the presence of a specific immune response to hFn14 (FIG.5). In this experiment, the Fn14 negative and positive cell populationswere mixed at a ratio of 1:1 prior to staining and analysis by flowcytometry. A subsequent negative result would be indicated by thepresence of either a fluorescence peak overlapping that of a negativecontrol, or a shift in the whole peak. The appearance of a double peakwould indicate a positive result (see positive control in panel A.iii.).Interestingly, only those mice immunized with live cells suspended inPBS were positive for antibodies recognizing cell surface expressedhFn14, whereas those immunized with antigen in adjuvant show nodetectable response (panel B). The spleens from 2 of these anti-hFn14positive mice were used for hybridoma fusion to generate monoclonalantibodies. This fusion yielded clones that recognize cell surfaceexpressed hFn14. Antibodies from three of these clones were purified andare designated CRCBT-06-005, CRCBT-06-006 and CRCBT-06-007. The isotypeof these monoclonal antibodies was assessed and CRCBT-06-005 andCRCBT-06-007 were found to be IgG2bκ and CRCBT-06-006 was found to beIgG2a.

Characterization of Monoclonal Antibodies to Human Fn14

Tweak Induced NFκB Blocking

Antibodies CRCBT-06-001, CRCBT-06-002, CRCBT-06-003, CRCBT-06-004,CRCBT-06-005, CRCBT-06-006 and CRCBT-06-007 were purified and assessedfor functional activity in vitro. All purified antibodies exhibitedTweak induced NFκB blocking activity as expected. CRCBT-06-001 andCRCBT-06-002 were able to fully block activation at 100 ng/ml (FIG. 6A).CRCBT-06-003 and CRCBT-06-004 also demonstrated good blocking ability.CRCBT-06-005, CRCBT-06-006 and CRCBT-06-007 efficiently blocked NFκBactivation by 200 ng/ml of Tweak-Fc when added at a concentration of 1μg/ml (FIG. 7A). At the lower antibody concentration of 100 ng/ml,blocking was also observed.

Antibodies ITEM-1 and ITEM-2 were also assessed for their ability toeither activate NFκB or to block Tweak induced NFκB in the reporterassay (FIGS. 7A and B). ITEM-1 was unable to activate NFκB, i.e., cellscultured in the presence of ITEM-1 display no difference in NFκBactivation as compared to cells alone. ITEM-1 also had no effect on theactivation of NFκB by Tweak-Fc. In contrast to CRCBT-06-001,CRCBT-06-002, CRCBT-06-003, CRCBT-06-004, CRCBT-06-005, CRCBT-06-006,CRCBT-06-007 and ITEM-1, ITEM-2 induced consistent NFκB activation.

CRCBT-06-001 and CRCBT-06-002 Functional Activity in Kym1 Cell DeathAssay

CRCBT-06-001, CRCBT-06-002, CRCBT-06-003 and CRCBT-06-004 were assessedfor the ability to block the Tweak induced cell death of Kym1 cells invitro. Cells alone display only a small degree of cell death and thispercentage increases when cells were cultured in the presence ofincreasing levels of Tweak-Fc (FIG. 8). When cells were co-cultured inthe presence of TweakR-Fc the Tweak-induced cell death was blocked.Similarly, when cells cultured in the presence of Tweak were co-culturedin the presence of CRCBT-06-001 or CRCBT-06-002 (1 μg/ml) the cellsremain viable indicating efficient blocking of Tweak-Fc induced celldeath. At a lower concentration (100 ng/ml) of antibody, Tweak inducedcell death was blocked at lower concentrations of Tweak-Fc. CRCBT-06-003and CRCBTG-06-004 also show good blocking of Tweak-induced Kym1 celldeath but not to the same level as for CRCBT-06-001 and CRCBT-06-002.

IL-8 Secretion Assays

CRCBT antibodies were assessed for functional activity in an in vitroIL-8 secretion assay. The properties of the CRCBT antibodies wereassessed on A375 cells by either determining if the CRCBT antibodiescould trigger IL-8 secretion (agonist) or inhibit the Tweak-Fc-inducedIL-8 secretion (antagonist).

A375 cells were cultured in the presence or absence of antibody at arange of concentrations (0, 0.01, 0.1, 1, 10 μg/ml; FIG. 9). Cells alonedisplayed no detectable background levels of IL-8. A series of isotypecontrols were assessed and displayed no IL-8 secretion. High IL-8secretion was seen in the presence of CRCBT-06-001 and CRCBT-06-006.This IL-8 secretion was specific as the levels increase in an antibodyconcentration dependent manner. Weak IL-8 secretion is observed in thepresence of CRCBT-06-002, CRCBT-06-004, CRCBT-06-005, CRCBT-06-007,ITEM-1 and ITEM-4 (FIG. 9).

Antibodies (10 μg/ml) were also assessed in the presence of 300 ng/mlTweak-Fc to determine antagonist properties (FIG. 10). High antagonistactivity was seen for CRCBT-06-002, CRCBT-06-005, CRCBT-06-007, ITEM-4comparable to an anti-Tweak blocking antibody. Weak antagonist activityis seen for CRCBT-06-004 comparable to an anti-Tweak blocking antibody(FIG. 10).

An equation was employed to estimate the level of agonist activity asfollows: (a−c)/(b−c). A similar calculation was used to estimate degreeof antagonist activity: (b−d)/(b−a). Where a=the amount of IL-8 secretedin the presence of 10 μg/ml antibody, b=the amount of IL-8 secreted inthe presence of 300 ng/ml Tweak-Fc, c=the amount of IL-8 secreted fromcells in the absence of either antibody or Tweak-Fc, d=the amount ofIL-8 secreted in the presence of 10 μg/ml antibody and 300 ng/mlTweak-Fc. Table 1 summarizes these calculations for all antibodiestested with exception of the antagonist activity for those antibodieswith strong agonist properties, as the calculation is not valid in thoseinstances.

TABLE 1 Calculated percentage agonist/antagonist activity in IL-8secretion assay % % Agonist Antagonist CRCBT-06-001 128 N/A CRCBT-06-00211 94 CRCBT-06-004 12 46 CRCBT-06-005 26 92 CRCBT-06-006 106 N/ACRCBT-06-007 16 89 ITEM-1 21  9 ITEM-4 8 97 IgG1 1 32 IgG2b 0 33 IgG2a 132 Anti-Tweak 0 99 Rat IgG1 1  3

Thus these data together with those from assays to determine NFκBsignaling and/or kym1 cell death indicate that antibodies have agonistactivity in some functional assays and antagonist activity in otherassays.

Affinity Measurement of Antibodies Using Fn14-Fc

A Bio-Rad ProteOn XPR36 was used to determine the affinity of mousemonoclonal anti-Fn14 antibodies CRCBT-06-001 and CRCBT-06-002 torecombinant Fn14-Fc. It is clear that both antibodies tested display ahigh on-rate and a low off-rate. The ProteOn software was used to fit acurve to the sensorgram based on a mathematical model, enabling thekinetics of the interaction (ka, Kd and K_(D)) to be calculated. Anumber of models can be used to fit the curves to the sensorgram,essentially the models used for each sensorgram were used as theyprovided the best fit, as evident by the low Chi² value obtained for theresiduals. Results are shown in Tables 2 and 3.

TABLE 2 Binding kinetics of CRCBT-06-001 for recombinant Fn14-Fc.Parameter ka Kd K_(D) Chi2 Units 1/Ms 1/s RU Scope Global Global AutoDefined Type Fitted Fitted Fitted N/A Kinetic- 2.36 × 10⁰⁴ 1.81 × 10⁻⁰⁵7.68 × 10⁻¹⁰ 6.66 Langmuir

TABLE 3 Binding kinetics of CRCBT-06-002 for recombinant Fn14-Fc.Parameter ka Kd K_(D) Chi2 Units 1/Ms 1/s RU Scope Global Global AutoDefined Type Fitted Fitted Fitted N/A Kinetic- 1.52 × 10⁴ 1.42 × 10⁻⁰⁵9.35 × 10⁻¹⁰ 4.97 Bivalent analyteAffinity Measurement of Antibodies Using Fn14

Binding kinetics of anti-Fn14 antibodies CRCBT-06-001, CRCBT-06-002,CRCBT-06-004, ITEM-1 and ITEM-4 were assessed as described herein andresults are set out in Table 4.

Of the selection of anti-Fn14 antibodies tested under identicalexperimental conditions as capture agents, CRCBT-06-004, ITEM-1 andITEM-4 displayed much faster dissociation compared to CRCBT-06-001 andCRCBT-06-002 (Table 4).

TABLE 4 Binding kinetics of anti-Fn14 antibodies for recombinant Fn14.Antibody ka Kd KD Units 1/Ms 1/s M CRCBT-06-001 1.86 × 10⁵ 1.02 × 10⁻⁴5.50 × 10⁻¹⁰ CRCBT-06-002 1.31 × 10⁵ 2.59 × 10⁻⁵ 1.98 × 10⁻¹⁰CRCBT-06-004 3.45 × 10⁵ 2.03 × 10⁻³ 5.88 × 10⁻⁹  ITEM-1 3.58 × 10⁵ 1.47× 10⁻³ 4.10 × 10⁻⁹  ITEM-4  5.1 × 10⁵ 8.64 × 10⁻⁴ 1.68 × 10⁻⁹ DNA Sequencing of Heavy and Light Chains of Antagonistic Antibodies

The light and heavy chains were sequenced for all antibodies thatantagonized NFκB signaling (FIG. 11). Subsets of antibodies share a highdegree of homology within their heavy and light chains with a number ofdifferences present.

Anti-hFn14 Antibodies Bind to a Conformational Epitope

To determine if the epitope bound by CRCBT-06-001 is conformational, theFn14-Fc purified protein was reduced and alkylated (R+A) to abolish thedisulphide bond structure. ELISA results using CRCBT-06-001 clearlydemonstrate that when the antigen is in the native folded state theantibody bound in a dose dependant manner. Upon reduction and alkylationhowever, this binding was abolished (FIG. 12Ai) demonstrating aconformational dependence for binding to CRCBT-06-001. Anti-human Fcrecognized the Fc portion of Fn14 indicating the integrity of R+A Fn14is retained (FIG. 12A ii). Western blot analysis also confirmed thisfinding (FIG. 12B).

ELISA was also used to determine if the epitope(s) bound byCRCBT-06-002, CRCBT-06-003, CRCBT-06-004, CRCBT-06-005, CRCBT-06-006,and CRCBT-06-007 is(are) conformationally dependent. Antibodies weretested at 1 μg/ml, and, as shown in FIG. 12C all antibodies bind to adisulfide dependant conformation of hFn14.

Generation of a Phage Library Expressing Random Fn14 Fragments

The coding region for Fn14 was amplified and digested with DNase I.Conditions that yielded fragments between 50 and 400 by were used toproduce a mixture of Fn14 gene fragments. The fragments were cloned intophagemid vector pHENH6 to create a gene fragment library with the sizeof ˜6×10⁶ independent clones. A number of randomly picked clones wereassessed and demonstrated the broad size distribution of the library.

Panning the Fn14 Gene Fragment Library with CRCBT-06-001

Four rounds of panning were performed to select gene fragments of Fn14which bound to CRCBT-06-001. An ELISA was performed and very highsignals were observed demonstrating enrichment in binding of genefragments displayed on phage to CRCBT-06-001 in rounds 1-4.

Individual clones were selected from round 4 panning, a PCR performedand the resulting DNA fragments were sequenced. The sequence identity ofround 4 selected Fn14 gene fragments is shown in FIG. 13, panel A.Fragments were re-assessed by ELISA for anti-hFn14 antibody bindingcapability (FIG. 13, panel B). Those fragments that bound comprised aportion of the extracellular region of Fn14 that contains all 6 cysteineresidues that form the 3 known disulfide bonds (RW114-RW131). Fragmentscontaining this region but also a portion of the transmembrane domaindid not appear to show specific binding in this experiment. Withoutbeing bound by theory or mode of action, these data may suggest thepresence of part of the transmembrane domain alters the structure orfolding of these fragments (RW95 & RW98). The shortest Fn14 fragmentidentified in the phage library panning experiments bound byCRCBT-06-001 is indicated in bold (RW129).

Construction of hFn14 Extracellular Domain Mutants and Sub-Domains to beExpressed on Phage

To further characterize the epitopes of the anti-Fn14 monoclonalantibodies, a panel of hFn14 constructs were designed to be displayed onphage. These include: The H. Sapiens Fn14 (hFn14) extracellular domain;four hFn14 extracellular domain mutants (D45A, K48A, M50A and D62E)known to have a decreased affinity for the natural ligand Tweak (Brownet al, 2006); and two sub-domains (1 and 2) delineated by disulfidebonds resolved in the NMR solution structure of the hFn14 extracellulardomain (He et al., 2009). Sub-domain 2 is truncated at the C-terminus,since panning the hFn14 gene fragment library on CRCBT-06-001 enrichedfor a fragment lacking the 7 C-terminal residues of the extracellulardomain, suggesting that these were not critical for antibody binding.The constructs cloned and expressed on phage are shown in FIG. 12A. Themutants illustrated in the context of the hFn14 structure homology modelderived by Brown et al., (2006) are shown in FIG. 14B.

hFn14 Extracellular domain Constructs are Expressed Successfully on theSurface of Phage

To adequately assess the relative amounts of each hFn14 constructexpressed on phage, a c-myc epitope tag was engineered between the pIIIminor coat protein of the phage and the displayed hFn14 construct. Thestrong reactivity of the anti-c-myc monoclonal antibody 9E10 (MAb 9E10)with the wild type hFn14 extracellular domain and the mutants indicatedthat these constructs expressed well on phage. When testedsimultaneously in the same ELISA, no significant binding of the phageconstructs to the negative control MAb 5G8 was observed, showing thatthe phage preparations bound specifically to MAb 9E10.

Phage Displayed Human Extracellular domain Mutants Show DecreasedBinding to Recombinant Tweak-Fc

The concentration of each phage preparation was adjusted to achieve anapproximately equal reactivity with MAb 9E10. This was performed tonormalize the amount of displayed hFn14 in each phage preparation forcomparison. When assessed by ELISA, each phage preparation was shown tohave an approximately equal reactivity with MAb 9E10, indicating thatthe preparations had been normalized successfully (FIG. 15, Panel A). Nosignificant reactivity to MAb 5G8 was observed in the same ELISA,indicating that binding was specifically to MAb 9E10 (FIG. 15, Panel B).To assess the relative reactivity of each hFn14 construct withrecombinant Tweak-Fc, normalized phage preparations were tested forbinding to the ligand by ELISA simultaneously (FIG. 15, Panel C). Incontrast to MAb 9E10, Tweak-Fc was most reactive with the wild-typehFn14 extracellular domain and less reactive with the four mutants (FIG.15, Panel C). No significant binding of Sub-domain 2 to Tweak-Fc wasobserved, suggesting that regions of the extracellular domain absent inthis construct contain residues critical for Tweak binding. Nosignificant binding of phage-displayed c-myc alone to Tweak-Fc orphage-displayed hFn14 to recombinant Fc alone was observed, suggestingthat the hFn14 constructs bound specifically to Tweak in these assays(FIG. 15, Panel D).

The Panel of Phage Displayed hFn14 Extracellular Domain Constructs Bindto Anti-Fn14 MAbs

The relative reactivity of each phage-displayed hFn14 construct toCRCBT-06-001, CRCBT-06-002, CRCBT-06-003 and CRCBT-06-004 anti-Fn14monoclonal antibodies was also assessed by ELISAs performed in parallel(FIG. 16A). In contrast to the pattern of reactivity observed withrecombinant Tweak-Fc, all the hFn14 mutant constructs, bound well to theanti-Fn14 MAbs CRCBT-06-001, CRCBT-06-002, CRCBT-06-003 andCRCBT-06-004. As shown in previous assays, no significant binding of thephage-displayed constructs to MAb 5G8 was observed, indicating that thephage constructs were binding specifically to the anti-Fn14 MAbs (FIG.16B). Furthermore, no significant binding of phage-displayed c-myc tagalone to any of the anti-Fn14 MAbs was observed, suggesting theantibodies were binding specifically to displayed Fn14 (FIG. 17).Binding of ITEM-1 to the 4 mutants was comparable to the binding towildtype Fn14 extracellular domain (FIG. 17).

Anti-Fn14 Monoclonal Antibodies Bind to Sub-Domain 2, but not Sub-Domain1 of the hFn14 Extracellular Domain

To further characterize the epitopes bound by anti-Fn14 monoclonalantibodies, CRCBT-06-001, CRCBT-06-002, CRCBT-06-003, CRCBT-06-004,CRCBT-06-005, CRCBT-06-006, CRCBT-06-007, ITEM-1 and ITEM-2 were testedfor binding to the phage-displayed hFn14 extracellular domain,sub-domain 1 and sub-domain 2 by ELISA (FIG. 18A, FIG. 19, Panel A).Phage were normalized according to their levels of reactivity with MAb9E10 assayed in parallel. Consistent with previous ELISA data,CRC-BT-06-001 and CRCBT-06-002 reacted well with both the full-lengthextracellular domain and sub-domain 2. Furthermore, CRC-BT-06-003 toCRCBT-06-007, ITEM-1 and ITEM-2 bound well to these phage-displayedhFn14 constructs. None of the anti-Fn14 MAbs bound to phage-displayedsub-domain 1, even though this construct bound well to MAb 9E10 whenassayed in parallel. As expected, none of the phage constructs bound tothe negative control antibody MAb 5G8 (FIG. 18B, FIG. 19B).

Binding of Antibodies to Fn14 Sub-Domains

As described above, residues included in the epitope(s) of anti-hFn14mAbs CRCBT-06-001, CRCBT-06-002, CRCBT-06-003, CRCBT-06-004,CRCBT-06-005, CRCBT-06-006 and CRCBT-06-007 have been shown to bind tosub-domain 2 and not to sub-domain 1 of hFn14 using phage display.Synthetic peptides corresponding to sub-domain 1p (a longer form ofsub-domain 1 expressed on phage) and 2 and a further smaller region ofhFn14 (termed sub-domain 3) were produced in order to confirm thesefindings (FIG. 20).

CRCBT-06-001, CRCBT-06-002, CRCBT-06-003, CRCBT-06-004, CRCBT-06-005,CRCBT-06-006, CRCBT-06-007 and ITEM-1 did not bind to sub-domain 1p(FIG. 21A) but were reactive with a synthetic peptide representingsub-domain 2 of Fn14 when compared to an isotype control antibody (FIG.21A). The order of reactivity from strongest to weakest wasCRCBT-06-002, CRCBT-06-001, CRCBT-06-005, CRCBT-06-007, ITEM-1 andCRCBT-06-006. The reactivity of CRCBT-06-001 and CRCBT-06-002 wassimilar and at 5 ng/ml (FIG. 21B). This point was in the linear part ofeach curve and was used to measure the percentage decrease in binding ofthe other mAbs to sub-domain 2. There was a 40% decrease in binding ofCRCBT-06-005, 48% decrease in binding of CRCBT-06-007, 68% decrease inbinding of ITEM-1 and a 76% decrease in binding of CRCBT-06-006 comparedto CRCBT-06-001 and CRCBT-06-002 binding to sub-domain 2. This suggestseither the affinity of CRCBT-06-001 and CRCBT-06-002 is greater for thisdomain, or the epitope could vary slightly for CRCBT-06-005 toCRCBT-06-007 and ITEM-1.

The reactivity of CRCBT-06-001 CRCBT-06-002, CRCBT-06-003, CRCBT-06-004,CRCBT-06-005, CRCBT-06-006 and CRCBT-06-007 with sub-domain 3 was lowerthan for sub-domain 2, indicating this region may be a partial epitopefor these antibodies. However, the differences in binding to this regioncould reveal subtle variations in the contact regions of the antibodieswith hFn14. The order of binding from strongest to weakest wasCRCBT-06-005, CRCBT-06-002, CRCBT-06-001, CRCBT-06-006, CRCBT-06-007 andITEM-1. The relative binding of each antibody to sub-domain 3 at asingle point in the linear region of the curves was measured at 0.2μg/ml (FIG. 21C). There was a 43% decrease in binding of CRCBT-06-002compared with CRCBT-06-005 for binding to sub-domain 3, 60% decrease forCRCBT-06-001, 73% decrease for CRCBT-06-006 and CRCBT-06-007 and a 76%decrease in binding of ITEM-1 to sub-domain 3 compared with CRCBT-06-005mAb. This could indicate the epitope of ITEM-1 may differ slightly tothe CRCBT antibodies based on weaker interaction with surface exposedresidues in sub-domain 3.

Fn14 Homologue Mutants Bind to Anti-Fn14 mAbs

A series of mutants were generated that represent single amino acidmutations that change the human Fn14 residue to the mouse and/or ratequivalent. These were designed for expression on phage. Mutantsincluded T33N (SEQ ID NO: 49), A34S (SEQ ID NO: 50), R38S (SEQ ID NO:51), R56P (SEQ ID NO: 52) and L77M (SEQ ID NO: 53). The relativereactivity of each phage-displayed Fn14 constructs to the antibodiesCRCBT-06-002 and ITEM-1 antibodies was assessed by ELISA. All the hFn14constructs bound well to CRCBT-06-002. The R56P mutant although slightlyreduced on CRCBT-06-002 displayed no binding to ITEM-1. Furthermore, nosignificant binding of phage-displayed c-myc alone to any of theanti-Fn14 mAbs was observed, suggesting the antibodies were bindingspecifically to the phage displayed Fn14 (FIG. 22).

As shown in previous assays, no significant binding of thephage-displayed constructs to a control mAb 5G8 was observed (FIG. 22),indicating that the phage constructs were binding specifically to theCRCBT-06-002 or ITEM-1 (FIG. 22).

Comparison of Antibody Binding to Human and Mouse Fn14 and Mutant HumanFn14

Wild type human and mouse Fn14 extracellular regions were cloned as Histagged fusion constructs for expression in bacteria. Proteins wereexpressed and purified to yield folded soluble protein. Assessment ofthe capability of each antibody to bind to human and mouse Fn14extracellular domain was assessed by ELISA (FIGS. 23A, B and C). Allantibodies were shown in this assay format to bind well to the wild typehuman Fn14 extracellular domain. The ability of antibodies to bind mouseFn14 revealed differences in antibody specificity. AntibodiesCRCBT-06-001, CRCBT-06-002, CRCBT-06-005, ITEM-2 and ITEM-4 all boundwell to mouse Fn14 whereas CRCBT-06-003 and ITEM-1 displayed reducedbinding.

Since the R56P mutant displayed no binding to ITEM-1 when expressed onphage (FIG. 22), this mutant was further assessed for binding to thesuite of CRCBT antibodies along with the commercial antibodies ITEM-1,ITEM-2 and ITEM-4. A number of R56 mutants were generated in order toassess the importance of this residue. His tagged fusion proteins weregenerated for mutant human Fn14 R56P, R56A (SEQ ID NO: 54) and R56K (SEQID NO: 55) extracellular region. These were expressed in bacteria andpurified to yield folded soluble protein. Assessment of the capabilityof each antibody to bind to this series of residue R56 mutants wasassessed by ELISA (results are depicted in FIG. 24).

Binding of antibody to the R56P mutant was seen for all antibodieshowever slightly reduced for CRCBT-06-003 and ITEM-1 (FIGS. 24A and B).To ensure the introduction of a proline residue was not simply alteringthe folding state of the protein, a similar mutant was assessed wherethe R56 was mutated to an alanine (R56A). Binding of antibody to thismutant mirrored that obtained for the R56P mutant (FIGS. 24C and D). Tofurther assess if the charged nature of the wild type arginine residuewas the reason that CRCBT-06-003 and ITEM-1 display a reduction inbinding, a R56K mutant was generated. The binding of CRCB-06-003 andITEM-1 was mostly restored suggesting that charge is important for theantigen-antibody interaction at this residue (FIGS. 24E and F).

In combination, the data generated for the R56 mutants displays thatalthough some reduced binding is present for CRCBT-06-003 on thismutant, all CRCBT antibodies do bind well to mutants of R56 andtherefore it can be concluded that it is not a critical residue in theantibody-antigen interaction.

A recombinant protein for the mutation R58A (SEQ ID NO: 56) was alsogenerated and the binding of all antibodies was assessed. The His-taggedfusion protein was expressed in bacteria and purified to yield foldedsoluble protein. Interestingly, this mutant segregated the antibodiesinto two groups, capable of binding or no binding (FIG. 25). Binding ofCRCBT-06-001, CRCBT-06-002, CRCBT-06-005 and ITEM-2 was unaffected bythe presence of this mutation, whereas CRCBT-06-003, ITEM-1 and ITEM-4binding is abolished.

Further analysis of the R58A mutant was performed using a phageexpressed version of the mutant. The binding of antibodies CRCBT-06-001and CRCBT-06-002 are again unaffected. ITEM-1 and ITEM-4 binding isabolished as previously and CRCBT-06-004 additionally is unable to bind(FIG. 26).

Alanine Scan of Subdomain 2

To determine specific residues within the Fn14 epitope that are criticalfor binding of each antibody, an alanine scan was performed across mostsub-domain 2 residues (with addition of a few residues outside thisregion). The suite of alanine mutants were generated as phage expressedmutants of the human Fn14 extracellular region.

The mutants assessed included W42A (SEQ ID NO: 57), L46A (SEQ ID NO:58), D51A (SEQ ID NO: 59), S54A (SEQ ID NO: 60), A57G (SEQ ID NO: 61),R58A (SEQ ID NO: 56), P59A (SEQ ID NO: 62), H60A (SEQ ID NO: 63), S61A(SEQ ID NO: 64), D62A (SEQ ID NO: 65), F63A (SEQ ID NO: 66) and L65A(SEQ ID NO: 67). Where an alanine residue was present in the Fn14 (as inthe case of A57) a glycine residue was used to generate the mutation.The binding of CRCBT-06-001, CRCBT-06-002 and CRCBT-06-004 along withITEM-1, ITEM-4, and in some cases ITEM-2 were assessed for binding toeach of the mutants (FIGS. 27 and 28). As expected, in all cases thecontrol antibody 5G8 did not display any binding to the phage expressedproteins. All anti-Fn14 antibodies assessed bound well to wild typehuman Fn14 (FIG. 27A). Antibody binding was not effected for any of theantibodies on mutants W42A (FIG. 27B), S54A (FIG. 27C), A57G (FIG. 27D),P59A (FIG. 27E), S61A (FIG. 27F), F63A (FIG. 27G) and L65A (FIG. 27H)suggesting these are not important residues for antibody-antigenbinding.

All antibodies bound to the L46A (FIG. 28A), D51A (FIG. 28B) and D62A(FIG. 28C) mutants however the binding of ITEM-4 is partly reduced ineach case.

All antibodies were able to bind the H60A mutant however a slightlyreduced binding is noted for CRCBT-06-004, ITEM-1 and ITEM-4 (FIG. 29A).This effect was not observed for CRCBT-06-004 and ITEM-1 when themutation was replaced with a lysine, however for ITEM-4 binding wasabolished (H60K; SEQ ID NO: 68; FIG. 29B). The binding of ITEM-2 to theH60K mutant vary from those in WO2009/140177 and may warrant furtherinvestigation.

Analysis of Antibody Binding Dependence on Individual Disulfide Bonds

The Fn14 extracellular region contains three disulfide bonds (cysteineresidues termed 1-6) of which two reside within sub-domain 2 (cysteineresidues 3-6; FIG. 20 panel C). As all the CRCBT antibodies bound wellto sub-domain 2 in the context of a peptide binding assay, the need forthe disulfide bonds was assessed one pair at a time. The cysteineresidues 3 and 6 were mutated to serine residues (FIG. 20, Panel E) in apeptide and binding of anti-Fn14 antibodies was assessed. This disulfidebond proved dispensible with all antibodies to Fn14 tested displayingbinding (FIG. 30A). The cysteine residues (4 and 5) that create thesecond disulfide bond were mutated to serine in a peptide of sub-domain2 (FIG. 20, Panel F). There was a clear difference in the reactivity tothis peptide with antibodies CRCBT-06-001, CRCBT-06-002 and ITEM-4displaying good binding, whereas binding for CRCBT-06-004, CRCBT-06-005and ITEM-1 is disrupted (FIG. 30B).

EXAMPLE 2 Treatment of a Cancer-Mediated Wasting Disorder with Anti-Fn14Antibodies

Many methods used in the present Example have been previously describedin Example 1. Some new methods are described in the following text,together with results of experiments.

Detection of hFn14 Expression in Cell Lines Used for Tumor Formation

To assess the level of hFn14 expression in cell lines used for in vivotumor formation studies, induced and un-induced tumor cell lines werestained using an anti-hFn14 specific antibody and assessed by flowcytometry. Cells containing inducible hFn14 display a high level ofinducible expression (FIG. 31, panel A. +4-OHT). Although in theun-induced state these cells should contain no expression of targetprotein, a small level of hFn14 is clearly evident (indicated by anarrow). The absence of this low level staining in the other cell linesassessed which do not contain hFn14 confirms the specificity of theantibody therefore confirming the expression in un-induced cells (FIG.31, panels B and C).

IL-6 Secretion by Tumor Cell Lines In Vitro

MEF tumor cell lines containing hFn14 or hFn14-GPI (induced or uninducedfor protein expression) were cultured for 48 hours under normal growthconditions, the media from cells was harvested and the levels of IL-6were assessed (FIG. 32). The levels of secreted IL-6 were significantlyincreased in hFn14 containing cells after induction with 4-OHT (FIG.32).

Establishment of hFn14 Tumor Model in Mice

An in vivo tumor model in wildtype C57BL/6 mice was created comprising4-OHT inducible hFn14. To create tumor cell lines, SV40 immortalized MEFcells were transformed with human v12Hras and then stably infected withan inducible hFn14 construct described in the Materials and MethodsSection of Example 1.

Mice were inoculated sub-cutaneously with cells (+/− inducible hFn14).Tumors began to form and were measurable by day 4 (FIG. 33A, panel A).The growth rate of these tumors was similar between hFn14 and controls,however the hFn14 tumors grew larger at later time points. Concurrently,from day 8 mice bearing hFn14 tumors rapidly suffered weight loss andtheir overall general health deteriorated (FIG. 33 B), with micedeveloping cachexia. All mice in this group were euthanized by day 10due to the presence of these symptoms. Interestingly, the tumors inthese mice were still in the un-induced state indicating that inducibleexpression in this system was not tightly regulated. On postmortemanalysis, increased vasculature was noted in the hFn14 tumors ascompared to the non-hFn14 control tumors.

Creation of Non-Signaling hFn14 Control Tumors

As the control tumors used in previous experiments with this systemsimply lacked expression of the target protein, a more suitable controlconstruct was created for use as a control. A ‘non-signaling’ hFn14 wascreated whereby the extracellular region of hFn14 was fused to theC-terminal glycosylphosphatidylinositol anchor (GPI) coding region fromTrailR3 to create hFn14-GPI (FIG. 34A). When expressed on cells, thisprotein would appear from the outside of the cell the same as wildtypehFn14 but would simply lack functional signaling. An equivalent tumorcell line harboring 4-OHT inducible GPI-anchored hFn14 was generatedusing this hFn14-GPI construct. Mice were injected with tumor cells onday 1 of the experiment and monitored for body weight, appearance oftumors and for general health throughout the experiment. The appearanceof tumors was evident in all mice by days 4 (FIG. 34B, panel i). Similarto the non-hFn14 tumors in the previous experiments the hFn14-GPI had aslightly lower growth rate past day 8 when compared to the hFn14 tumors.In hFn14-GPI tumor mice, no weight loss or other signs of physicalillness (i.e., cachexia) was noted and to ensure these mice would notsimply develop symptoms at a later time point, mice were observed up to30 days. The hFn14-GPI tumors grew large and had no effect on theoverall health of the mice when compared to that seen for hFn14 tumors(FIG. 34B, panel ii). Upon postmortem assessment, hFn14-GPI controltumor vascularization appeared much like the non-expressing controls inthe previous experiments.

Constitutive Fn14 Expressing Tumors

As the level of hFn14 in tumors was low and therefore difficult todetect, and given the inducible feature of this model was not beingused, another method to demonstrate that wildtype hFn14 was responsiblefor the increased vasculature and onset of physical symptoms wasgenerated. An independent cell line was generated with constitutiveexpression of hFn14. The expression of hFn14 in these cell lines wasverified. A tumor experiment was performed comparing theseconstitutively expressing tumor cells to the previous hFn14 tumors (FIG.35). Tumors formed in both groups however constitutively expressinghFn14 tumors did not show any signs of cachexia (i.e., weight loss andphysical illness) around days 8-12 (as seen for the previous hFn14tumors). These mice were observed over time and did eventually exhibitcachexia symptoms from approximately day 18. This result indirectlyvalidated the involvement of hFn14 in the weight loss and deteriorationof health seen when wildtype hFn14 is present in tumors.

Antibody Treatment of hFn14 Tumors

The treatment of hFn14 tumors with blocking antibodies to Fn14 was nextassessed in the inducible tumor model with low level Fn14 expression.Mice were inoculated with hFn14 or hFn14-GPI tumor cells. The size oftumors, weight and overall health of mice was assessed daily. As theonset and severity of cachexia symptoms (i.e., weight loss and declininggeneral health) is swift in this model, mice were first treated with theblocking antibody CRCBT-06-001 (10 mg/kg) 2 days prior to the expectedonset of weight loss (FIG. 36). Prior to this initial antibodyadministration, hFn14 and hFn14-GPI groups were randomly segregated into+/− antibody treatment groups. Treatment was continued twice a week fora total of 4 weeks. Control mice received no treatment. The hFn14-GPItumor group remained healthy as predicted throughout the course of theexperiment, with the antibody treated group maintaining good generalhealth. A small decrease in average weight was noted in the antibodytreated group however mice appeared otherwise healthy. As expected, theuntreated hFn14 group exhibited weight loss and deterioration of generalhealth from day 8. All mice were euthanized by day 12. hFn14 mice thatreceived antibody treatment not only maintained body weight and overallhealth throughout the course of the treatment but survived an overall of15-25 days longer than untreated mice. Results are depicted in FIG. 36.

Assessment and Comparison of CRCBT-06-001, CRCBT-06-002, CRCBT-06-004and ITEM-1 Efficacy

Antibodies CRCBT-06-001, CRCBT-06-002, CRCBT-06-004 and ITEM-1 were invivo in a single dose administration experiment (FIG. 37). hFn14expressing tumors were formed in mice and on day 7 when the onset ofweight loss was on average apparent, a single IP dose of 5 mg/kgantibody was administered to groups of mice. A group of hFn14 tumor micereceived no treatment. As expected, CRCBT-06-001 was able to rescue themajority of mice in the group. CRCBT-06-002 rescued all mice with aprompt reversal of weight loss was evident as early as 48 hours afterantibody administration. Administration of antibodies CRCBT-06-001 andCRCBT-06-002 lead to increased survival time of 17.9 and 22.3 days,respectively (see FIG. 37B; however in both cases at least one mouseremained healthy and maintained body weight at the termination of thestudy at day 27 meaning that the recited day is likely anunderestimate). Untreated mice displayed no effect on illnessprogression and demonstrated a survival time of about 10.3 days. Micetreated with ITEM-1 or CRCBT-06-004 had an average survival time of 11days and 10 days, respectively. These data demonstrate that CRCBT-06-001and CRCBT-06-002 provide a superior benefit in treating cachexiasymptoms and extending life in this mouse model.

Histopathology of Tumors

Histopathology was performed on the tumors from mice bearing hFn14 orhFn14-GPI tumors as well as a surviving CRCBT-06-002 antibody treatedhFn14 tumor bearing mouse. The hFn14-GPI expressing tumor formed adiscrete entity where the muscle-tumor interface was clearly defined. Incontrast, hFn14 expressing tumor cells invaded the surrounding skeletalmuscle and a clear boundary between tumor cells and muscle could not beidentified. In mice bearing an hFn14 expressing tumor that had beentreated with antibody CRCBT-06-002 and subsequently survived until day32, no invasion of the surrounding skeletal muscle by the tumors cellswas apparent, with a clear tumor-muscle interface being visiblecomparable to that seen for the hFn14-GPI tumor.

CRCBT-06-001 Dose Response and Rescue Treatment

As twice weekly treatment with CRCBT-06-001 at a high dose was sosuccessful, mice were next treated with a single injection of antibodyat a range of doses (0-10 mg/kg) to assess the smallest effective amountof antibody required to observe an effect (FIG. 38). Untreated miceagain suffered the expected fate however in this case the onset ofweight loss (i.e., cachexia) began at day 7. Mice in the 10 mg/kg groupfollowed the same pattern as before with a dramatic increase in overallhealth and maintenance of body weight compared to the untreated group.The onset of weight loss was apparent in 2 mice in this group at days 14and 16. The other mouse survived healthy to the end of the experiment(23 days). In the 5 mg/kg treated group, all mice remained healthy andmaintained weight throughout the course of the experiment with a smalldecrease in weight becoming apparent around day 21-22 suggesting theclearance of the antibody from the body. This dose was overall moreeffective than the higher dose of 10 mg/kg.

All other antibody treated groups (0.1, 0.5 and 1 mg/kg) showed somedegree of improvement post treatment but these lower doses only delayedthe onset of weight loss (i.e., cachexia) by a few days, with a declinein body weight seen between days 8-10. For the mice in these groups,when the onset of cachexia symptoms was definite (days 9 or 10) a singleantibody dose of 10 mg/kg was administered to assess the possible rescueof these mice from further decline. The 0.5 and 1 mg/kg groups werefully rescued from further weight loss and surprisingly displayed a fullreversal of weight loss within approximately 5 days post treatment. Thisreversal was maintained throughout the remainder of the experiment (upto day 23).

EXAMPLE 3 Treatment of a Cancer-Mediated Wasting Disorder with Anti-Fn14Antibodies

Methods

Experimental Animals

Female C57BL/6 mice (11 week old) were given a single subcutaneousinjection of mouse embryonic fibroblasts (MEF s) stably transfected withFn14 (MEF Fn14) or the untransfected MEF Hras cells (Hras) (cell linegeneration described in the Materials and Methods Section of Example1.). On day 6, mice were given a single intraperitoneal injection ofIgG2b isotype control antibody (Hras+IgG2b, MEF Fn14+IgG2b, n=8/group)or CRCBT-06-001 (MEF Fn14+001, n=8). Mice were housed under a 12:12-hourlight-dark cycle. Mice were assessed daily for general health, bodymass, tumor size, food and water intake.

Grip Strength Test

Whole body strength was assessed on day 11 by means of a grip strengthmeter (Columbus Instruments, Columbus, Ohio), essentially as describedin Murphy et al., 2012.

Assessment of Functional Properties of Tibialis Anterior Muscles In Situ

On day 11, mice were anaesthetised with sodium pentobarbitone (Nembutal;60 mg/kg; Sigma-Aldrich) via intraperitoneal (IP) injection. The methodsfor assessment of the contractile properties of the mouse tibialisanterior (TA) muscle in situ have been described previously (Murphy etal., 2010). After determining peak tetanic force, muscles were subjectedto a 4 minute intermittent stimulation protocol to induce musclefatigue. Muscles were maximally stimulated for 1 second every 4 secondsfor the duration of the fatigue protocol. Peak tetanic force wasassessed at 5 minutes and 10 minutes following cessation of thefatiguing stimulation protocol. At the conclusion of the contractilemeasurements in situ, the TA, extensor digitorum longus (EDL), soleus,plantaris, gastrocnemius and quadriceps muscles as well as theepididymal fat and heart were carefully excised, blotted on filter paperand weighed on an analytical balance. Mice were killed as a consequenceof heart excision while still anaesthetised deeply.

Skeletal Muscle Histology

Serial sections (5 μm) were cut transversely through the TA muscle usinga refrigerated (−20° C.) cryostat (CTI Cryostat; IEC, Needham Heights,Mass.). Sections were reacted with: laminin (#L9393, Sigma-Aldrich) fordetermination of mean myofibre cross-sectional area (CSA); succinatedehydrogenase (SDH) to determine activity of oxidative enzymes; andN2.261 (developed by Dr. Helen M. Blau, obtained from the DevelopmentalStudies Hybridoma Bank developed under the auspices of the NICHD andmaintained by The University of Iowa, Department of Biology, Iowa City,Iowa, USA) to assess the percentage of myosin Ha isoforms (Murphy etal., 2010). Mouse TA muscle have been shown previously to almost lacktype I fibers (Murphy et al., 2011) so all non-N2.261 reacting fiberswere assumed to represent type IIx/b fibers. Optical density (o.d.) ofSDH was determined after 6 min of reactivity for all samples andsections were captured in full color using bright field light microscopyand analyzed, substantially as described in Murphy et al., 2010. Digitalimages were obtained using an upright microscope with camera (AxioImager D1, Carl Zeiss, Wrek, Gottingen, Germany), controlled andquantified by AxioVision AC software (AxioVision AC Rel. 4.7.1, CarlZeiss).

Results

Injection with MEF Fn14 caused a loss of body mass, with IgG2b treatedmice losing ˜20% body mass during the 11 day experimental periodcompared with control mice that only lost ˜3% body mass (FIG. 39).However, treatment with CRCBT-06-001 attenuated the loss of body masswith MEF Fn14-001 treated mice only losing ˜5.5% body mass (FIG. 39).Tumor mass and volume was not significantly different between groups(FIG. 40).

MEF Fn14 injection caused a loss of muscle and fat mass, with a 15-29%decrease in mass of the plantaris and tibialis anterior (TA) muscles anda 60% decrease in subscapular fat mass compared with control mice (FIG.41). The loss of muscle and fat mass in MEF Fn14 injected mice wascompletely prevented by a single injection of CRCBT-06-001 (FIG. 41B).

Given the short duration of the experiment, there were no differencesbetween groups in assessments of muscle function, with similar gripstrength (FIG. 42) and peak twitch and tetanic force of TA muscles(FIGS. 43A and 43B). When TA muscle strength was examined over a rangeof stimulation frequencies, there was a main effect for lower tetanicforce production in MEF Fn14+IgG2b treated mice compared with controls(FIG. 43C). During a 4-minute intermittent fatiguing stimulationprotocol, TA muscles from MEF Fn14+IgG2b treated mice produced higherforces than controls (FIG. 43D).

TA muscle sections stained with haematoxylin and eosin and sectionsreacted with an anti-laminin antibody revealed that MEF Fn14 injectioncaused a decrease in cross-sectional area of type IIa fibres (−21%) andtype IIx/b fibres (−13%), and a 13% decrease in average cross-sectionalarea. However, a single injection of CRCBT-06-001 completely preventedthe decrease in muscle fibre size. There were no differences betweengroups in the proportion or oxidative enzyme capacity of type IIa andtype IIx/b fibres (FIG. 44).

EXAMPLE 4 Treatment of a Colon Cancer-Mediated Wasting Disorder withAnti-Fn14 Antibodies

Method

Male CD2F1 (11 week old) or female Balb/c (9-10 week old) mice weresub-cutaneously (s/c) inoculated with 1×10⁶ Colon-26 cells on day 1(Cell Line Services; Germany). Mice were assessed daily for generalhealth, body mass, food and water intake. Tumors were measured daily andtumor volume was calculated based on (1*w²)/2 where 1=length andw=width. For pair fed groups feeding was achieved by monitoring the foodintake of the untreated tumor group each 24 hours and then providing anon-tumor ‘pair-fed’ group with that amount of food for the following 24hour period.

Results

Assessment of Antibodies in Colon-26 Mouse Cancer Model of Cachexia

The Colon-26 cancer model of cachexia is known in the cachexialiterature (Murphy et al., 2012). CD2F1 mice were inoculated withColon-26 cells to form subcutaneous solid tumors. These tumors lead toprogressive weight loss in mice which includes the wasting of skeletalmuscle.

To assess the effect of anti-Fn14 antibody in this model,Colon-26-bearing mice were treated with IP antibody CRCBT-06-002(Colon-26+CRCBT-06-002) prior to the onset of weight loss. Multipledosing was performed throughout the course of the experiment asindicated in FIG. 45A. Additional control groups included a pair-fednon-tumor group (Pair Fed), an untreated tumor group (Colon-26Untreated) and an untreated non-tumor group (Control). Efficacy ofCRCBT-06-002 was demonstrated with treated mice maintaining body massand overall good health throughout the experiment (longest time pointmeasured was day 27) when compared to untreated mice that all reachedthe maximum allowed weight loss (based on institutional animal ethicsapproval) by days 14-18 (FIGS. 45A and 45B). Note, two of the fiveantibody treated mice were killed on day 20 due to maximal tumor mass(based on the institutional animal ethics approval) being reached andnot due to weight loss or other signs of illness.

Assessment of Antibodies in Colon-26 Tumor-Bearing Balb/c Mice

Colon-26 tumors are also commonly formed in Balb/c mice and given theeffect of CRCBT-06-002 that had been noted in CD2F1 mice, a similartreatment schedule was carried out in Balb/c mice bearing Colon-26tumors. Results are depicted in FIG. 46 and show that treatment withCRCBT-06-002 reduces tumor size.

Assessment of Antibody CRCBT-06-001 in Colon-26 Tumors in CD2F1 Mice

Given the efficacy demonstrated by CRCBT-06-002 on Colon-26 cancercachexia, the effect of CRCBT-06-001 on cachexia in this model was nextassessed. CD2F1 mice were inoculated with Colon-26 cells and mice weretreated with 10 mg/kg IP antibody CRCBT-06-001 on days 16 and 20 asindicted. Untreated mice were injected with Colon-26 cells but receivedno antibody treatment. Mice treated with CRCBT-06-001 displayed adelayed onset of weight loss as compared to the untreated controls,demonstrating the effectiveness of the antibody on the cachexiaprogression in this model (FIG. 47A). This can also be seen onassessment of survival over time (FIG. 47B). Despite the effectivenessof the antibody in preventing weight loss, it did not appear to have asignificant effect on tumor volume (FIG. 47C).

EXAMPLE 5 Treatment of a Colon Cancer-Mediated Wasting Disorder withAnti-Fn14 Antibodies

Methods

Experimental Animals

Male CD2F1 mice (12 week old) were inoculated subcutaneously (s/c) with5×10⁵ Colon-26 (C-26) cells (kindly donated by Martha Belury, The OhioState University, Columbus, Ohio) suspended in 100 μl of sterilised PBS.Mice were assessed daily for general health, body mass, tumor size, foodand water intake. All mice were obtained from the Animal ResourcesCentre (Canning Vale, Western Australia) and housed in the BiologicalResearch Facility at The University of Melbourne under a 12:12-hourlight-dark cycle. Water was available ad libitum and both water andstandard laboratory chow was provided, changed and monitored daily. Pairfeeding was achieved by monitoring the food intake of the untreatedgroup each 24 hours and then providing the pair-fed group with thatamount of food for the following 24 hour period.

Grip Strength and Rotarod Test

Whole body strength and whole body mobility and coordination wereassessed on day 21 by means of a grip strength meter (ColumbusInstruments, Columbus, Ohio) and rotarod performance test (Rotamex-5,Columbus Instruments) essentially as described in Murphy et al., 2012.

Assessment of Functional Properties of Tibialis Anterior Muscles In Situ

On day 22, mice were anaesthetised with sodium pentobarbitone (Nembutal;60 mg/kg; Sigma-Aldrich) via intraperitoneal (IP) injection. The methodsfor assessment of the contractile properties of the mouse tibialisanterior (TA) muscle in situ have been described in detail previously(Murphy et al., 2010). After determining peak tetanic force, muscleswere subjected to a 4 minute intermittent stimulation protocol to inducemuscle fatigue. Muscles were maximally stimulated for 1 second every 4seconds for the duration of the fatigue protocol. Peak tetanic force wasassessed at 5 minutes and 10 minutes following cessation of thefatiguing stimulation protocol. At the conclusion of the contractilemeasurements in situ, the TA, extensor digitorum longus (EDL), soleus,plantaris, gastrocnemius and quadriceps muscles as well as theepididymal fat and heart were carefully excised, blotted on filter paperand weighed on an analytical balance. Mice were killed as a consequenceof heart excision while still anaesthetised deeply.

Skeletal Muscle Histology

Skeletal muscle histology was assessed substantially as described inExample 3.

Results

Assessment of Antibodies in Colon-Mouse Cancer Model of Cachexia

To further assess the effect of anti-Fn14 antibody in the Colon-26model, mice were inoculated with Colon-26 cells and prior to the onsetof weight loss, treated with antibody CRCBT-06-002 (C-26+002). Multipledosing was performed throughout the course of the experiment asindicated. Control groups included an untreated tumor group (C-26) and atumor group injected with IgG antibody (C-26+IgG). The C-26+IgG andC-26+002 groups were pair-fed to the untreated tumor group.

Clear efficacy of CRCBT-06-002 is demonstrated with treated micemaintaining body mass and overall good health throughout the experiment(longest time point measured was day 22) when compared to untreated miceand IgG treated mice (FIGS. 48A and B). Over the 22 day period,untreated and IgG treated tumor mice lost ˜29% tumor-free body mass,whereas CRCBT-06-002 treated tumor mice only lost ˜4% tumor-free bodymass (FIG. 48C). Tumors were measured over time (FIG. 49A) and tumorvolume was calculated and graphed for each group (FIGS. 49B and 49C).

Absolute mass of the extensor digitorum longus (EDL), plantaris,tibialis anterior (TA), gastrocnemius and quadriceps muscles were 23-34%higher in the CRCBT-06-002 treated tumor mice compared to the untreatedand IgG treated mice (FIG. 50A). Fat and heart mass were also higherwith CRCBT-06-002 treatment (FIG. 50B).

Peak grip strength was 35% and 26% higher in CRCBT-06-002 treated tumormice compared with untreated and IgG treated mice, respectively (FIG.51A), whereas latency-to-fall during the rotarod test was notsignificantly different between groups (FIG. 51B).

TA muscles from CRCBT-06-002 treated tumor mice were stronger thanuntreated and IgG treated mice, with higher peak twitch force andtetanic force over a range of stimulation frequencies (FIGS. 52A, 52Band 52C). During a 4-minute intermittent fatiguing stimulation protocol,TA muscles from 002 treated mice were stronger than untreated and IgGtreated tumor mice (FIG. 52D).

TA muscle sections stained with haematoxylin and eosin and sectionsreacted with an anti-laminin antibody revealed that CRCBT-06-002 treatedmice have bigger muscle fibres than untreated and IgG treated mice. Theproportion of type IIa muscle fibres appears similar between groups, butthe area of the type IIa fibres is larger in the CRCBT-06-002 treatedmice. Muscle fibre oxidative capacity as assessed by SDH reaction wassimilar between groups.

Analysis of TA muscle sections stained with haematoxylin and eosin andsections reacted with an anti-laminin antibody revealed thatcross-sectional area (CSA) of type IIa and type IIx/b fibres as well asaverage fibre CSA was greater in CRCBT-06-002 treated mice thanuntreated and IgG treated mice (FIG. 53A). The proportion of type IIaand type IIx/b muscle fibres was similar between groups (FIG. 53B).Oxidative enzyme capacity in type IIa and type IIx/b fibres as well asaverage fibre oxidative capacity as assessed by SDH reaction was similarbetween groups (FIG. 53C). These data indicate that treatment withCRCBT-06-002 increased the size of muscle fibres but did not change theconstitution of muscles compared with controls.

EXAMPLE 6 Treatment of Diabetes and a Diabetes-Mediated Wasting Disorderwith Anti-Fn14 Antibodies

Methods

Diabetes was induced in 8 weeks old male C57Bl/6 mice by administeringmultiple low doses of Streptozotocin (STZ) essentially as described inChen et al., 2009 and/or Motyl et al., 2009. Briefly, mice received anIP injection of 45 mg/kg STZ (Sigma S-0130) for 5 consecutive days (days1-5). Control mice were injected with vehicle (0.1M chilled sodiumcitrate buffer, pH4.5). Mice were assessed for general health, bodymass, food and water intake daily. The pair-fed (PF) group was fedaccording to the STZ injected group daily intake.

For antibody treatment, mice were administered a single IP 20 mg/kg doseof CRCBT-06-002 two days after the final streptozotocin injection (day7). On day 28 all mice were sacrificed and blood, muscle (tibialisanterior, quadricep and heart) and epididymal fat was removed foranalysis. Non-fasted blood glucose levels were measured using anAccu-check blood glucose monitor.

Results

A STZ-induced diabetes mouse model was used to assess the effect ofantibody CRCBT-06-002 on diabetes-associated weight loss. Antibody wasadministered on day 7 to a group of STZ induced diabetic mice. Body masswas assessed daily and standardised against starting body mass as 100%.Standardised group averages were graphed (FIG. 54). The administrationof antibody after diabetes induction lead to an immediate drop in bodymass of this group however this weight was recovered well by 12-14 days.An overall increase in body mass was then seen from around day 18 ascompared to the STZ alone group.

Over the course of the 28 day experiment, group daily intake of waterwas measured and graphed as the cumulative intake (FIG. 55). STZ micewater intake was higher due to the diabetic state, however thepost-antibody treatment STZ group display a water intake trend over timecloser to the intake of the non-diabetic control groups.

On day 28, mice were euthanized and blood glucose levels were assessed(FIG. 56). When a blood glucose level out of range of the test wasobtained, the maximum level of detection was assigned (600 mg/dl). Thiswas the case for three of the STZ group and one of the antibody treatedgroup.

Epididymal fat, heart, tibialis anterior (TA) and quadricep were removedfor analysis. Group average tissue weight was calculated and graphed(FIG. 57A) standardised to final body mass (day 28; FIG. 57B) andagainst starting body mass (day 1; FIG. 57C). The antibody treatmentattenuated the decrease in quadriceps and heart, and partiallyattenuated the decrease in TA muscle and in epididymal fat.

These data demonstrate that treatment with an anti-Fn14 antibody(CRCBT-06-002) prevents or treats diabetes-induced cachexia and reducessymptoms of diabetes, such as increased daily water intake and bloodglucose levels.

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We claim:
 1. An isolated Fn14-binding monoclonal antibody or antigenbinding fragment thereof, the antibody or fragment comprising: (i) aheavy chain variable region (V_(H)) comprising the sequence set forth inSEQ ID NO: 16 or a humanized, chimeric, synhumanized or deimmunizedversion thereof, wherein the humanized, chimeric, synhumanized ordeimmunized version thereof comprises a V_(H) comprising CDRs 1, 2 and 3of a V_(H) comprising the sequence set forth in SEQ ID NO: 16; and (ii)a light chain variable region (V_(L)) comprising the sequence set forthin SEQ ID NO: 23 or a humanized, chimeric, synhumanized or deimmunizedversion thereof, wherein the humanized, chimeric, synhumanized ordeimmunized version thereof comprises a V_(L) comprising CDRs 1, 2 and 3of a V_(L) comprising the sequence set forth in SEQ ID NO:
 23. 2. Anisolated Fn14-binding monoclonal antibody or antigen binding fragmentthereof, the antibody or fragment comprising: (a) a heavy chain variableregion (V_(H)) comprising the sequence set forth in SEQ ID NO: 16 or ahumanized, chimeric, synhumanized or deimmunized version thereof,wherein the humanized, chimeric, synhumanized or deimmunized versionthereof comprises a V_(H) comprising CDRs 1, 2 and 3 of a V_(H)comprising the sequence set forth in SEQ ID NO: 16; and (b) a lightchain variable region (V_(H)) comprising the sequence set forth in SEQID NO: 23 or a humanized, chimeric, synhumanized or deimmunized versionthereof, wherein the humanized, chimeric, synhumanized or deimmunizedversion thereof comprises a V_(L) comprising CDRs 1, 2 and 3 of a V_(L)comprising the sequence set forth in SEQ ID NO: 23 which has one or moreof the following characteristics: (i) does not detectably bind to apolypeptide comprising a region of Fn14, the region consisting of asequence set forth in SEQ ID NO: 33 or 46; (ii) binds to anextracellular domain of Fn14 comprising a proline or alanine substitutedfor the arginine at position 56 of SEQ ID NO: 1 at a similar orsubstantially the same level as it binds to an extracellular domain ofFn14; (iii) binds to an extracellular domain of Fn14 comprising analanine substituted for the arginine at position 58 of SEQ ID NO: 1 at asimilar or substantially the same level as it binds to an extracellulardomain of Fn14; (iv) binds to an extracellular domain of Fn14 comprisingan alanine substituted for the histidine at position 60 of SEQ ID NO: 1at a similar or substantially the same level as it binds to anextracellular domain of Fn14; (v) binds to a peptide consisting of thesequence set forth in SEQ ID NO: 48 (optionally with an additional sixhistidine residues at a terminus) at a similar or substantially the samelevel as it binds to an extracellular domain of Fn14; (vi) binds to anextracellular domain of Fn14 comprising an alanine substituted for thetryptophan at position 42 of SEQ ID NO: 1 at a similar or substantiallythe same level as it binds to an extracellular domain of Fn14; (vii)binds to an extracellular domain of Fn14 comprising one of the followingamino acid substitutions (numbering relative to SEQ ID NO: 1) T33N,A34S, R38S, R56P, L77M, R56A, R56K, R58A, W42A, L46A, D51A, S54A, A57G,P59A, H60A, S61A, D62A, F63A, L65A or H60K; (viii) binds to aconformational epitope in Fn14; and/or (ix) does not detectably bind toa polypeptide comprising an extracellular domain of Fn14 fused to an Fcregion of an antibody, wherein the polypeptide is reduced or reduced andalkylated.
 3. A monoclonal antibody that binds specifically to Fn14 andcomprises at least a heavy chain variable region (V_(H)) and a lightchain variable region (V_(L)), wherein the V_(H) and V_(L) bind to forma Fv comprising the antigen binding domain, and wherein the Fv comprisesa V_(H) comprising CDRs 1, 2, and 3 of a V_(H) comprising the sequenceset forth in SEQ ID NO: 16 and a V_(L) comprising CDRs 1, 2, and 3 of aV_(L) comprising the sequence set forth in SEQ ID NO:
 23. 4. Themonoclonal antibody or antigen binding fragment thereof of claim 1,wherein the V_(H) and the V_(L) are in a single polypeptide chain andthe Fn14-binding protein is: (i) a single chain Fv fragment (scFv); (ii)a dimeric scFv (di-scFv); (iii) at least one of (i) and/or (ii) linkedto a heavy chain constant region or an Fc or a heavy chain constantdomain (CH) 2 and/or CH3; or (iv) at least one of (i) and/or (ii) linkedto a protein that binds to an immune effector cell; or wherein the V_(L)and V_(H) are in separate polypeptide chains and the Fn14-bindingprotein is: (a) a diabody; (b) a triabody; (c) a tetrabody; (d) a Fab;(e) a F(ab')₂; (f) a Fv; (g) at least one of (a) to (f) linked to aheavy chain constant region or an Fc or a heavy chain constant domain(CH) 2 and/or CH3; (h) at least one of (a) to (f) linked to a proteinthat binds to an immune effector cell; or an antibody.
 5. The monoclonalantibody or antigen binding fragment thereof of claim 1, which is ananti-Fn14 antibody, the antibody comprising a heavy chain variableregion (V_(H)) comprising the sequence set forth in SEQ ID NO: 16 and alight chain variable region (V_(L)) comprising the sequence set forth inSEQ ID NO:
 23. 6. A composition comprising the monoclonal antibody orantigen binding fragment thereof of claim 1 and a suitable carrier.